The Stillway Guide to Flying

The Stillway Guide to Flying By Bill Stillway, Ph.D. For MUSC Students of Molecular Basis of Medicinei Before you is a metabolic map, over which you about to fly. At Your present altitude it is difficult for you to see many details, because it has been almost two years since you took off in your metabolic airplane (This was originally written as a study aid for students preparing for Part I of the National Boards, but at the request of new students, it is now made available to everyone.). It is time to make some practice landings in various metabolic locations and refresh your memory. You have probably noticed that many details are omitted on the map, because I have assumed that you have studied these subjects at one time in detail and need only to be reminded of a few things you may have temporarily forgotten. Get your text, syllabus, notes, etc. gathered and ready. We will go through each of the pathways step-by-step. You will need to look some things up. The purpose here is to help you recall facts and to think metabolically. Our engines are warmed up, flaps are down, and we are ready for take-off. Standardized tests, such as the National Boards, are now asking more problem-solving questions. You therefore are expected to use metabolic information to interpret and solve medical problems. For example, if asked what would accumulate if the enzyme pyruvate kinase is catalytically defective in red blood cells, you should know that phosphoenolpyruvate is the answer. Further, you should be able to say something about the overall metabolic consequences of such a defect. To answer this question, you must know that pyruvate kinase is a glycolytic enzyme and that it converts phosphoenolpyruvate to pyruvate. Secondly, you need to know that the main function of glycolysis in the red blood cell is the generation of 2,3-bisphosphoglycerate (BPG), which is required for the regulation of oxygen binding to hemoglobin. Since BPG decreases oxygen affinity, it will accumulate in a pyruvate kinase deficiency, and oxygen affinity of hemoglobin will be severely decreased. Before we begin this review, keep in mind that the map is designed around the liver, but that other tissues are also covered within the text of this review. Additionally, the cell membrane and organelles other than the mitochondrion are omitted. You will also have to think about the whole body and cell types on a physiological and cell biological level. Now, bring your engines to full-throttle; we are taking off! Please observe that this document is copyrighted and cannot be reproduced, sold or put on the internet without the expressed permission of the author. Whereas, it has been offered free of charge, some have taken this document as their own and placed it on hand-held computers, as well as the internet without permission. Don’t cheapen yourself as a plagiarist. How to use the map. Steps are annotated by number on the metabolic map. The number '"1" refers to the interconversion of glucose and glucose 6-phosphate. i Department of Biochemistry & Molecular Biology Medical University of South Carolina 173 Ashley Ave. P.O. Box 250509 Charleston, SC 29425 Copyright, L. W. Stillway Revised 2005 Guide to Flying 1 Find number 1 and land your plane. 1. Glucose and glucose 6-phosphate 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 Name three enzymes involved in the intracellular interconversion of glc and G-1-P (accounting for all cells). How do the reactions differ thermodynamically? Which of the two kinases is allosterically regulated, and name the effector (modifier)? The liver contains an enzyme associated with the conversion glc  G-6-P. Name it and state the significance of the higher Km it has compared to that of its isozyme found in other tissues. What is the effect of insulin on the transport of glc into cells of the liver, skeletal muscle, brain, and adipose tissue? (That is, in which of these tissues is glucose transport insulin independent?) How does the phosphorylation of glucose to G-6-P affect its transport out of cells? Is phosphorylation necessary for the transport of glucose into cells? Is glucose transport an example of active transport? How would one determine whether ATP or G-6-P contains a high-energy phosphate? The binding of insulin to its receptor results in the phosphorylation of what amino acid residue? (There are three choices -- serine, threonine and tyrosine.) Which one of these is most often phosphorylated when covalent modifications of enzyme proteins occurs, as in glycogen metabolism? Glucose is transported into the liver cell by which transporter? Which other cells are insulin independent with respect to glucose transport? Which cells are insulin dependent with respect to glucose transport? Name this transporter. 1.10 1.11 1.12 You are at the end of the runway, but a few multiple-choice questions have appeared before you, but the tower has instructed that you cannot take off until they are answered and understood. Throughout, questions will appear occasionally. You will find answers by consulting the explanations given in the boxes, but do try to answer the question first by marking your answer and try to understand the concept so that you will be able to answer other questions about the same subject. All of the following enzymes catalyze reactions that directly generate ATP or GTP except which one? A. Malic enzyme B. Succinyl CoA synthase C. Phosphoglycerate kinase D. Pyruvate kinase E. ATP synthase Generally, reactions involving ATP or GTP are catalyzed by kinases. Recall that the malic enzyme is not a kinase, and it generates NADPH. Succinyl CoA synthase generates GTP from GDP, a substrate-level phosphorylation in the TCA cycle. Phosphoglycerate kinase and pyruvate kinase generate ATP in glycolysis and are substrate-level phosphorylations. All of the following stimulate liver fructose 1,6-bisphosphatase activity (directly or indirectly) except which one? A. Fructose 2,6-bisphosphate B. ATP C. Citrate Copyright, L. W. Stillway Revised 2005 Guide to Flying 2 Fructose 2,6-bisphosphate inhibits liver fructose 1,6-bisphosphatase (FBPase-1) and stimulates PFK-1. ATP and citrate allosterically stimulate FBPase-1. Glucagon and epinephrine stimulate FBPase-1 indirectly by stimulating the production of cAMP, which results in phosphorylation (covalent modification) of PFK-2-FBPase-2 (a tandem enzyme). This inhibits PFK-2 activity and stimulates FBPase-2 activity, by lowering the concentration of fructose 2,6-bisphosphate. A lower [fructose 2,6bisphosphate] stimulates FBPase activity and lowers PFK-1 activity. It is now time to take off again and head east to 2. Glycogen You will probably want to refer to a more detailed diagram of glycogen metabolism in a textbook for these questions. The land of glycogen metabolism is punctuated with many concepts. For example: whereas the main function of liver glycogen is to buffer blood glucose, skeletal muscle glycogen functions as quick fuel during early muscle contraction and cannot be used to buffer blood glucose, because muscle lacks the enzyme glucose 6-phosphatase. 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 What are the most important regulatory enzymes involved in glycogenolysis? What is the main regulatory enzyme associated with glycogenesis? Increased [cAMP] results in the phosphorylation of what three enzymes involved in glycogen metabolism? Which tissue contains the greatest concentration of glycogen? Which tissue contains the greatest whole-body store of glycogen? What two types of glycosidic linkages does glycogen contain? How is this similar to plant starch. What are the amylopectin and amylose portions of plant starch? Explain why cellulose is not digested by most animals, but starch and glycogen are easily digested. Name 4 disaccharidases normally found in the brush border of intestinal mucosal cells. Which one of the disaccharidases is most often lacking in man? What is the name of the disorder? Name the common intermediate between glycogenesis and glycogenolysis. What is the metabolic defect in glycogen storage disease type I? Suppose that debranching enzyme is defective. What would one expect to see in terms of glycogen structure? Glycogen is isolated from the liver of an individual with a metabolic problem. The ratio of glucose 1-phosphate to free glucose is 100:1. Which enzyme is most likely defective? What is the activated glucose intermediate in glycogenesis? What’s this? You have just taken off and are headed east again to 3, but the windshield is blinded by a huge piece of paper that says: Phosphorylase b is an enzyme that A. catalyzes the addition of glucose residues (as UDP-glucose to glycogen). B. cleaves phosphate esters in phosphorylated proteins. C. utilizes Pi as a substrate. D. directly requires ATP to convert glycogen to glucose 1-phosphate. E. contains a calmodulin subunit which is stimulated by increased [Ca2+]. Copyright, L. W. Stillway Revised 2005 Guide to Flying 3 Phosphorylase basically exists in two forms, phosphorylase b (inactive, non-phosphorylated) and phosphorylase a (active, phosphorylated). Although the b form is considered inactive, it can be allosterically modified to an active form (It is still phosphorylase b, because it has not been phosphorylated to phosphorylase a). Phosphorylase, whether a or b, requires Pi as a substrate to cleave a glucose residue from a non-reducing end of glycogen. Glucose 1-phosphate is produced and may be reversibly converted to glucose 6-phosphate by phosphoglucomutase. Note that the cleavage of one glucose residue, as glucose 1-phosphate, from glycogen can result in the net synthesis of three high-energy phosphate bonds (3ATP) in anaerobic glycolysis. One free glucose molecule can result in the net production of only two high-energy phosphate bonds in anaerobic glycolysis, because one ATP is consumed to produce glucose 6phosphate. Glycogen is isolated from the livers of two different infants. The glycogen is digested with the appropriate cofactors, phosphorylase and the debranching enzyme. Glycogen from individual A produces glucose 1-phosphate and free glucose in a ratio of 10:1. Glycogen from individual B produces a ratio of 1000:1. Which one of the following is most true of the result? A. Individual B has a normal glycogen structure. B. The glycogen in A contains less α(16) linkages. C. The glycogen in B contains more α(14) linkages and has glycogen storage disease type IV. D. Individual B is likely to be suffering from von Gierke's disease. E. Individual B most likely has a defective debranching enzyme. In tissues, glycogen is degraded metabolically by the combined action of phosphorylase and debranching enzyme, a tandem enzyme with two activities in the same peptide chain. Phosphorylase produces glucose 1-phosphate. Debranching enzyme produces free glucose. Since normal glycogen has a branch an average of every 10 glucose residues, and it will produce 10 glucose 1-phosphates for every free glucose. Glucose 1-phosphate is produced from α(14) linkages and free glucose from (16) linkages. Since α(16) linkages are at branches, the ratio of G-1-P to free glucose indicates the average number of glucose residues between branches. Individual A has normal glycogen structure. Individual B has a glycogen structure with very low branching and probably has a defective branching enzyme (Debranching enzyme is most likely OK.). His glycogen has many more α(14) linkages and fewer α(16) linkages than normal and has defective branching enzyme (Anderson’s disease). Von Gierke's is caused by defective glucose 6phosphatase. Which of the following contains β-glycosidic linkages? A. Plant starch B. Amylopectin C. Pectin D. Skeletal muscle glycogen E. Cellulose Cellulose contains linear chains of β(14) linkages. It is not digestible in mammals, because they lack the appropriate cellulases that hydrolyze β-glycosidic linkages. Ruminants and wood-eating insects have bacteria and protozoa in their GI tracts that produce cellulases.) Plant starch contains two components, pectin (linear) and amylopectin (branched). All of the glycosidic linkages are α(14) and α(16). Skeletal muscle glycogen has the same structure as liver glycogen, and they both resemble the amylopectin portion of starch, which has fewer branches than glycogen. Copyright, L. W. Stillway Revised 2005 Guide to Flying 4 3. Galactose metabolism You have been rocking your plane, and finally, the questions fly off the windshield when you take a sudden dive. You are lost, but as you head straight down you see a large “3” painted on the rooftop of a dairy barn. Coming out of your dive, there is a runway right before you, and you land with a few bounces and pull up next to a tall silo. The diary farmer dressed in coverall approaches your plane and says, “Yup, I know why you are here. I get this same plane every year, and I have some dairy questions for you. Shut your engine down and come on in to my milking parlor.” 3.1 “I am sure you know that the major source of galactose in the diet is diary products like the ones I produce here. 3.2 Now, tell me, what is the name of milk sugar and what are its constituents? 3.3 Some people cannot consume dairy products because of enzyme defects involved in the metabolism of galactose. What is the name of this disorder? 3.4 I guess you know that there are several variants of this disorder, and depending on which one an infant has, the severity will be much different. Can you name the enzymes that are defective in two of the major types? 3.5 Can you tell me what is the significance of UDP-glucose in the metabolism of galactose? Here, I have a blackboard just waiting to be written on. Show me the pathway. 3.6 Hey, I bet you could even show me a pathway for lactose synthesis. Y’know, those cows out there in the field are making lactose the same way humans do. 3.7 Good. Before you take off in that beautiful plane, there is one last thing I want you to remember without fail. What clinical sign in an infant is indicative of galactosemia? Lactose from the diet is hydrolyzed A. by the enzyme lactase. B. in the intestine. C. in the brush border of intestinal mucosal cells. D. by water. E. to galactose and glucose, which are transported to the liver via the portal vein. F. All of the above are true. Lactose is one of the many disaccharides that must be digested in the gut. All of the disaccharidases are located in the brush-border of the intestinal mucosal cells. These enzymes hydrolyze the disaccharides, such as sucrose, maltose, isomaltose and lactose to their individual monosaccharide units, which are transported via the portal vein to the liver. All hydrolytic reactions require water to split a covalent linkage. In these cases, it is splitting a glycosidic linkage, which is otherwise known as an acetal. Lactase is the most sensitive of the disaccharidases to intestinal insults, such as inflammation or infection. Temporary lactase deficiency is especially common in the young. Here, the other disaccharidases are still present and the easiest treatment is to remove all dairy products from the diet until the intestine is healed. Older adults and some ethnic groups, such as those of African or Asian descent have a rather high incidence of lactase deficiency. In these cases, the enzyme is not expressed by the cells. If deficient, galactokinase A. results in lower-than-normal blood levels of galactose. B. usually results in an accumulation of galactitol in the lens and formation of cataracts in infants. C. is the most severe form of galactosemia. D. catalyzes the conversion of galactose to galactose 6-phosphate. E. usually results in the accumulation of galactose 1-phosphate in the liver and sorbitol in the lens. Copyright, L. W. Stillway Revised 2005 Guide to Flying 5 The most common physical sign of galactosemia is the detection of cataracts in infants. This is a often definitive sign of galactosemia. The two most common types involve defective galactokinase and uridyl transferase. Defective uridyl transferase is the most severe form. One of the postulated reasons for this is that galactose 1-phosphate accumulates in the liver, depriving it of inorganic phosphate, which is required for many reactions. Liver failure and mental retardation result in this form, but not with defective galactokinase. How is lactose made in the mammary gland? Well, this one is easy. First, UDP-glucose is made from G-1P (remember glycogen synthesis?). UDP-glucose is epimerized to UDP-galactose by a 4-epimerase. In the last step, glucose displaces the UDP, condensing with galactose to make lactose. “OK, it is milking time, so get in your plane and head for your next destination. Hey, did you know that milk protein has a very biological value? This means that it contains virtually all of the essential amino acids and in the proper proportions, but cow’s milk is deficient in copper, so be sure that babies who a fed cow’s milk get their proper mineral supplements. Remember now, you are going to have to cross the Glycolytic River to get there, so head west.” This is a short runway and the wind is from the north, so you head into the wind rev your engine to its maximum RPM and release the brakes. You pull up and just clear the trees at the end of the runway, bank left and head west. Below, you can see the Glycolytic River to which you are going to return soon. You can see enzymes all line up and intermediates quickly being converted to others as they are passed on from enzyme to another. According to your speed and time, you should be near “4.” It is cloudy, but between clouds you clearly see “4” etched into a cornfield, and you wonder how was such a large letter done so neatly? Circling, you see that there are cornfields as far as you can see and below is a runway with several small planes and a Leer Jet parked at one end. You line up perfectly with the runway and land, raising a cloud of dust and pieces or corn stalks. A large sign painted on the side of the hanger says, “Welcome to Maize Farms, producer of fine corn products. We sweeten the lives of good people.” 4. Fructose metabolism 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 Name the major carbohydrates in corn. The disaccharide in corn contains which monosaccharide units? Is it a reducing sugar? What is “high fructose corn syrup”? How is fructose handled metabolically differently from glucose? Hint. Does fructose affect insulin and leptin levels differently than glucose? Name the major metabolic problems that appear with the disorder fructose intolerance. How would you treat fructose intolerance? How can one metabolically explain hypoglycemia seen in fructose intolerance? Is fructose a reducing sugar? What is the difference between levulose and dextrose? Unlike the last farmer in coveralls, this farmer sports a highly tailored Italian suit and exits from his four story brick mansion with a surrounding porch graced with glistening white columns that are at least 16 feet tall and 3 feet in diameter at the feet. He pushes his hand into your’s and pumps with exuberant energy and a tight grip. “I am so glad you came to visit Maize farms, my name is Frank Maize.” “I am SC, a medical student from South Carolina, how big is your farm, Mr. Maize”? “Oh, it’s kinda hard to tell, I let my supervisor worry about all those details, but it is somewhere Copyright, L. W. Stillway Revised 2005 Guide to Flying 6 around 10,000 acres of nothing but corn.” “What kinds of things do you make from corn, Mr. Maize?” “Oh, you would never imagine, SC. We make everything from corn starch, silage, corn syrup to white lightning.” “White lighting! I thought that was illegal.” “Well, SC, I was just getting your attention, so to speak. You see, we have a huge fermentor and distillery over there in the large white building where we make ethanol as a fuel additive, and we also produce our brand of white lightning, called ‘Maize’s Grace’. It is all perfectly legal, you know, we pay our taxes.” “On your sign it says ‘We sweeten the lives of good people.’ What does that mean?” “Well, SC, we also produce one of the newest money makers in the world, high fructose corn syrup. Yes Siree, you see that Leer Jet over there? That was bought with HFCS.” “Tell me about HFCS.” “Sure thing, you see that green building over there? It is the largest we have, and that is where we produce HFCS. It’s a good thing that there is a soft-drink machine in every school, college, supermarket and office building, because it is making people like me a super billionaire.” “How do you make it?” “Well, it is quite simple, and I don’t know why no one thought of it before, but essentially, we use the enzyme invertase to hydrolyze the sucrose from the corn to convert sucrose to fructose and glucose. (Invertase is also called sucrase.) This increases the fructose concentration, and it not only makes the corn syrup much sweeter, but also gives it some other properties that are better for food production. Our best preparation increases the percentage of fructose to 55% of the sugars.” Metabolically, the first product of fructose in the liver is which one? A. Fructose 6-phosphate B. Fructose 1-phosphate C. Glucose 6-phosphate D. Glucose 1-phosphate E. Fructose 1,6-bisphosphate Fructose is transported into the intestinal mucosal cell with a specific transporter, then to the liver, where it is phosphorylated to fructose 1-phosphate by fructokinase. Defective fructose 1-phosphate aldolase results in all of the following EXCEPT which one? A. Hypophosphatemia B. Hypoglycemia C. Hepatomegaly D. Inhibition of fructose 1,6-bisphosphate aldolase. E. Hyperinsulinemia. It is interesting to note that the use of high-fructose corn syrup has exploded in the food industry. Unfortunately, the long-term effects of this product, when used as a sweetening agent are not known with certainty in humans, although there seems to be a correlation with animal models, where it has clearly been shown that there is a link between the ingestion of high-fructose corn syrup and glucose intolerance, insulin insensitivity, diabetes type 2, obesity and hypertriglyceridemia, etc. Fructose does not stimulate the secretion of insulin as does glucose, and it also does not affect the production of leptins. The corn industry claims that there is no relationship between high-fructose corn syrup and these metabolic disorders and further makes the statement that fructose and glucose are metabolized in the same way. Which one of the following best describes fructose? A. A ketose B. A six-carbon sugar C. An epimer of glucose D. A ketohexose isomer of glucose E. An optical isomer of glucose Copyright, L. W. Stillway Revised 2005 Guide to Flying 7 Glyceraldehyde and dihydroxyacetone phosphate are produced by the catalytic action of fructose 1phosphate aldolase, a reverse aldol condensation. What is the main metabolic fate of glyceraldehyde? A. It is excreted in the urine. B. It is phosphorylated to glyceraldehyde 3-phosphate by glyceraldehydes 3-phosphate. C. It is isomerized to dihydroxyacetone and phosphorylated by dihydroxyacetone kinase. D. It enters the hexose monophosphate shunt where it may be used to make NADPH E. It condenses with erythrose 4-phosphate, which is converted to sedoheptulose 7-phosphate. “Do you know about the Glycolytic River, Mr. Maize?” “Oh yes, we pay close attention to that, because a major part of this farm is the production of glyceraldehydes and dihydroxyacetone phosphate from fructose 1-phosphate. You see, we have to do this to energize the system with carbons from fructose. This whole farm is run by ATP. Now, take that farm down the road, the DNA company that sent them defective fructose 1-phosphte aldolase, and the farm is about to fold because of the accumulation of fructose 1-phosphate. They can’t get rid of it, and there isn’t enough inorganic phosphate for the metabolic machinery upon which we all depend. Hey, SC, maybe you can figure out a way to fix that DNA factory.” SC, now you can borrow my Leer jet and fly on to the glycolytic river. Look for a place where G-6P is being diverted into several branches, especially the HMP shunt. As you cross the glycolytic river, you can see that G-6-P is rapidly drawn into several paths. First, you see that in the liver, it may be hydrolyzed to free glucose by glucose 6-phosphatase, which is expressed only in the liver and kidney. Some of it continues down-river to fructose 6-phosphate, but now you see that G-6-P and NADP are bound by the enzyme glucose 6-phosphate dehydrogenase. Remarkably, this enzyme spits out NADPH and 6-phosphogluconolactone. You can see that NADPH is liberated by another enzyme and that carbons are also being contributed and used by the HMP to and from the Glycolytic river. We will land here to get more details. 5. Hexose monophosphate shunt (Pentose phosphate pathway and Phosphogluconate pathway) 5.1 5.2 5.3 5.4 5.5 5.6 5.7 What are the two primary functions of the HMP shunt? What other pathways are fed by products of the HMP shunt? What are the two reactions in which NADPH is synthesized? Name the enzymes. Which of these enzymes, when defective, may result in drug-induced hemolytic anemia? Which carbon of glucose is lost in the process of making ribulose 5-P? What are some of the fates of carbons from glucose in the HMP shunt? Why would the activity of the HMP shunt be greater in tissues like liver, adipose tissue and adrenals? What are the reversible and irreversible parts of the HMP shunt? What if NADPH is not metabolically required, but ribose 5-phosphate is needed for nucleotide biosynthesis. Where would the carbons of ribose 5-phosphate be derived? From the list below, mark the items that are either components or characteristics of the hexose monophosphate shunt. A. Synthesizes ATP B. Produces NADPH, which is used in reductive biosynthesis. C. May produce fructose 6-phosphate D. May produce glucose 6-phosphate E. Generates carbon dioxide F. Generates pentoses, such as ribose 5-phosphate. G. Is tied directly to the TCA cycle H. Consumes NADH produced in glycolysis Copyright, L. W. Stillway Revised 2005 Guide to Flying 8 Is regulated by [NADP] HMP enzymes are found in greater abundance where steroids and/or fat are produced in the body. K. One of its enzymes, glucose 6-phosphate dehydrogenase, when defective, may result in lifethreatening anemia when certain drugs are administered. L. Directly furnishes an intermediate necessary for the synthesis of DNA. The two main functions of the HMP shunt are to produce NADPH and pentose phosphates, such as ribose 5-phosphate, which is required for the synthesis of nucleotides, including ATP, NAD, NADP, CoA, FAD, etc. Although glucose is oxidized, no ATP is produced. Instead, NADPH is generated so that it can be used in reductive biosynthesis. Examples would include fatty acid and steroid biosynthesis. This is why the shunt is more active in the liver, adrenals and fat cells. Many of the mixed-function oxidases require NADPH. The HMP shunt is tied directly to glycolysis through glucose 6-P, glyceraldehyde 3-P and fructose 6-P. It cannot directly make glucose 6-P, because the reaction catalyzed by glucose 6-phosphate dehydrogenase is irreversible. In fact, both oxidative steps are irreversible. Some individuals have defective glucose 6-phosphate dehydrogenase, which normally goes undetected, but some drugs will inhibit the already defective enzyme, and too little NADPH is produced in red blood cells. NADPH is needed to maintain the correct ratio of reduced and oxidized glutathione. If glutathione becomes too oxidized, the RBC membrane is disrupted and hemolysis results. The defective enzyme in the Wernicke-Korsakoff syndrome is transketolase, and it does require TPP, which may be low in alcoholism. Since the defective enzyme cannot bind enough TPP, the syndrome may result. An old wizened woman dressed in a long, flowing blue gown approaches you as you stand on the bank of the Glycolytic river. She says, “Hi, my name is Georgia Gatekeeper. I can show you some neat stuff if you would like.” “Let’s go for it, my name is SC.” “Yes, I know, I have been waiting for you. Have you noticed how rapidly the Glycolytic river flows? In muscle, the enzymes are very abundant so that it can generate a lot of ATP in a short period of time. Notice, how the intermediates are flying by so fast that it is hard to follow them, and see how close together are the enzymes? I am sure you know that there is much more organization to metabolism than those studying it, and we have many more secrets waiting to be revealed. What you see on paper, like that metabolic map you have in your hands, doesn’t resemble at all what you are actually seeing, because we are standing in a three-dimensional network.” Pointing with her right hand, she says, “Now, you see that enzyme up there? It is phosphoglucose isomerase. Note that it is reversibly converting G-6-P and F-6-P. It is regulated by mass action. See, G-6-P is piling up and now it rapidly converts it to F-6-P.” Now her hand points to the pool of F-6-P and a much larger enzyme with many subunits. “Laughing loudly, she says, “Now, that PFK-1 had people fooled for many years. First, it was not known to be regulated. Then it was thought that it was allosterically regulated only by levels of citric acid, ATP and AMP. After that, someone noticed an undiscovered sugar phosphate accumulated in cells when glycolysis was activated. This turned out to be fructose 2,6-bisphosphate, and it is know thought to be the main regulator. It overrides the previously discovered allosteric modifiers. Now, the secret is out that it is also regulated by induction and repression, as well as degradation.” I tried to get a word in edgewise, but she was so excited, I couldn’t get her to stop talking. “Look, look, over there! A brand new PFK-1 is being synthesized on that ribosome over there. See, it is folding into its native state, which is determined by the primary structure of the protein. Remarkable, isn’t it. It will replace this one pretty soon. Now, turn around and take a look. Ubiquitin is in the process of breaking up a carcass of PFK-1 into smaller pieces. Now I have some questions for you. Oh, by the way, I think you know your way around now. You are on your own.” I. J. Copyright, L. W. Stillway Revised 2005 Guide to Flying 9 AMP v no AMP 0.5 1.0 [ATP] 6. Phosphofructokinase (PFK) This enzyme is specifically known as PFK-1. If you see the names PFK or FBPase, it is safe to assume that these are PFK-1 and FBPase-1. 6.1 6.2 6.3 6.4 6.5 Name the two enzymes here that are thought to constitute the major control point in glycolysis. Are the reactions reversible? Why? What are the reactions? Name the major effectors (modifiers) involved with these enzymes. Specify whether they are negative or positive effectors. Under what metabolic circumstances would they be effective? Explain how ATP can be both a negative effector and a substrate of PFK-1. How does citrate inhibit PFK-1? Where would citrate originate? What compound does PFK-2 make from fructose 6-P? Specify if this compound is a negative or a positive modifier of PFK-1 and of fructose 1,6-bisphosphatase? Tests covering glycolysis would not be complete without a few questions about the main regulatory step involving PFK-1 and fructose 1,6-bisphosphatase. [A low hig h[ AT [F-6-P] P] v TP ] . Consider the Michaelis plot of PFK-1. Note that velocity is plotted vs. the substrate fructose 6-P. The other substrate, of course, is ATP (see below). 1. 2. 3. 4. 5. The curve at high [ATP] represents what type of regulation? The shape of the curve is said to be what? This is an indication of what type of binding? What is the effect of higher concentrations of ATP on Vm and Km? What is cooperativity with respect to proteins? Does is occur with regard to non-enzyme proteins such as Hb and Mb? Consider the Michaelis plot above where the substrate is ATP. 1. 2. 3. 4. 5. What is the effect of increasing [ATP] up to 0.5 mM? How does [ATP] effect enzyme activity at higher concentrations? What effect does AMP have on enzyme activity? Does it change Vm or Km? Suppose that ATP binds to both a catalytic and allosteric site, how might AMP stimulate enzyme activity? Guide to Flying Copyright, L. W. Stillway Revised 2005 10 Glucagon secretion results in all of the following except which one? A. Activation of fructose 1,6-bisphosphatase by increased levels of fructose 2,6-bisphosphate. B. Activation of hormone sensitive lipase by covalent modification of the enzyme protein. C. Inactivation of pyruvate kinase by phosphorylation of the enzyme protein. D. Inactivation of phosphofructokinase by lack of fructose 2,6-bisphosphate. This is a difficult question, because of the amount of information and reasoning needed to derive the correct answer. For our purposes, glucagon acts by stimulating the cAMP cascade by binding to receptors in liver and fat cells, but not in skeletal muscle. Two important actions of glucagon are (1) to elevate blood glucose levels by stimulating glycogenolysis and gluconeogenesis in liver and (2) to elevate blood free fatty acids by stimulating lipolysis in fat cells by stimulating hormone sensitive lipase. Glucagon results in the activation of hormone sensitive lipase through phosphorylation of the enzyme protein, so choice B is true. Since glucagon diminishes the concentration of F-2,6-BP and stimulates gluconeogenesis, choice A is false and choice C is true. Pyruvate kinase is inactivated during gluconeogenesis by phosphorylation of the enzyme protein, so choice D is true. From the curves shown below, which of the following is most true? A. B. C. D. E. ATP is a typical competitive inhibitor of the enzyme. AMP is a substrate for the enzyme. ATP is a substrate for the enzyme. AMP relieves the inhibition of ATP. This is an example of classical Michaelis kinetics. The most correct answer is D, because ATP is both a substrate and inhibitor of the enzyme (PFK-1), so C is true but incomplete. PFK-1 has both a catalytic and allosteric site for ATP. It is thought that AMP stimulates PFK-1 by displacing ATP from an allosteric site, relieving the inhibition its inhibition. This is not an example of classical Michaelis kinetics, because the curves are sigmoidal. 7. The triose phosphate level of glycolysis 7.1 7.2 7.3 We are now at the triose phosphate-level of glycolysis. Name the two triose phosphates. Have any high-energy bonds been created so far? How many would have been used if one started with glucose, fructose, glycogen or galactose? 7.4 As you know, glyceraldehyde 3-P (G-3-P) may now be converted to pyruvate. You can easily see some of the major sources of glyceraldehyde 3-P on the map. Like G-6-P, this is a major branch point in glycolysis. What are some of the sources and fates of G-3-P? 7.5 The conversion of G-3-P to 1,3-diphosphoglycerate represents the first oxidation in glycolysis. Note the structures. An aldehyde is converted to an acid that is linked with Copyright, L. W. Stillway Guide to Flying 11 Revised 2005 7.6 7.7 7.8 7.9 phosphate. Now, whenever two acidic groups are linked together, what is the compound called? Does the resulting compound have a higher or lower free energy of hydrolysis than an ester? What is the source of phosphate in this reaction? If the concentration of phosphate were low, what effect would this have on glycolysis? Obviously, NADH is generated in this reaction. It is easy to see that NAD is required for the reaction to take place, because it is a substrate. It is therefore important to be able to regenerate NAD from NADH. How might this be done? The reduction of NAD+ is written NAD+ + XH2  NADH + H+ + X, because A. B. C. D. E. the reaction involves a transfer of a single electron and proton. NAD is only partially reduced. the phosphates in NAD are ionizable groups. a hydride ion is transferred. the free energy change is positive. Because of the unique structure of NAD and NADP, these molecules can accept two electrons but only one proton from another molecule. The extra proton is free in the medium. 8. 8.1 Glycerol 3-phosphate shuttle The glycerol 3-phosphate shuttle will be taken up later. 9. 9.1. 9.2. 9.3. 9.4. Phosphoglycerate kinase Now, you should know this reaction. What is accomplished at this point? What functional group provides enough free energy to make this conversion favorable? How many net ATP have been generated so far from one glucose? If 32P (as Pi) is introduced at reaction 7, where will it be found after reaction 9? The standard free energy change for the conversion of 1,3-bisphosphoglycerate to ATP is -4.5 kcal/mol and the free energy change under typical physiological conditions is +0.3 kcal/mol. Considering these thermodynamic measurements, which of the following is most true? A. The actual free energy change is -4.2 kcal/mol. B. The reaction is readily reversible under physiological conditions. C. Another enzyme is required to by-pass the reaction during glucogenesis. D. The reaction is spontaneous under physiological conditions. E. The reaction is typical of a pyrophosphate cleavage. The actual free energy change of a physiological reaction is usually different from the standard free energy change because of concentration and temperature differences. In this case, under physiological conditions, the apparent equilibrium constant is slightly less than one, which means that the reaction non-spontaneous, endergonic or unfavorable (whichever term you prefer), but because the free energy change is close to 0 (and the equilibrium constant is near 1), the reaction is readily reversible. Copyright, L. W. Stillway Revised 2005 Guide to Flying 12 Which of the following contains an anhydride linkage? O A. B. C. CH3 CH2 CH2 C SCoA O CH3 CH3 CH2 CH2 CH2 CH2 CH2 O O O- C CH2 O CH3 D. O CH2 CH2 O P OO O C CH2 CH3 E. H C HC H2C O OH O O P OO- An anhydride linkage is formed when two acidic groups link together with a loss of water. An ester linkage is formed when an alcoholic group links with an acidic group with a loss of water. D is the correct answer, because it contains a mixed anhydride linkage like that of 1,3-bisphosphoglycerate, where phosphoryl group is linked to a carboxyl group. It also contains an ester linkage. Can you locate both? Which other compound contains a high-energy linkage? A is correct, because it is a thioester. It is the breakage of thioester linkages that thermodynamically drive the TCA cycle in two locations. 10. Pyruvate kinase 10.1. 10.2. 10.3. 10.4. 10.5. 10.6. Getting to 10 requires several steps. One of them utilizes a mutase enzyme. Where did we encounter a mutase earlier? The mechanisms are completely analogous. (Check the phosphoglucomutase mechanism.) What is PEP? How is it classified metabolically? Now, write the reaction taking place at 10. Name the enzyme. Is the reaction reversible? Guide to Flying Copyright, L. W. Stillway Revised 2005 13 10.7. 10.8. 10.9. 10.10. 10.11. 10.12. 10.13. 10.14. 10.15. 10.16. 10.17. This enzyme is an important regulatory enzyme. How is it regulated? Covalent modification? Allosteric modification? How does covalent modification take place, i.e., what reactions take place? Under what physiological circumstances would one expect this enzyme to be covalently modified? Note that this enzyme follows the generation of PEP from TCA cycle intermediates. Might this have something to do with regulation of gluconeogenesis? Would one expect to see increased or decreased activity of pyruvate kinase when gluconeogenesis is stimulated? Why? In terms of energy, what is the significance of this reaction? Now that pyruvate has been generated, the glycolytic pathway has been completed. How many net high-energy phosphate bonds may be produced from one each of the following? • • • • • • Fructose Glucose One glucose residue from glycogen (as a product of phosphorylase) Galactose Glycerol Glyceraldehyde 3-P 10.18 10.19 10.20 How many net moles of high-energy phosphate are consumed in the conversion of glucose to pyruvate? Is the rate of glycolysis more or less rapid under anaerobic conditions? Which reactions of glycolysis are irreversible? (There are three.) Which one of the following enzymes catalyzes an irreversible step in glycolysis? A. Enolase B. Phosphoglyceromutase C. Phosphoglycerate kinase D. Pyruvate kinase E. Lactate dehydrogenase This is a classical glycolytic question. There are three irreversible steps in glycolysis. You need to know them and their enzymes [1. Hexokinase (glucokinase in liver), 2. Phosphofructokinase, and 3. Pyruvate kinase.} Note that they are all kinases. One glycolytic kinase, phosphoglycerate kinase, catalyzes a reversible reaction. Know this exception. 11. Lactate dehydrogenase 11.1 11.2 11.3 11.4 11.5 11.6 Recall earlier (reaction 7) that NADH was generated. In order for glycolysis to continue, it must have some mechanism for regenerating NAD. Name the enzyme involved at 11 that regenerates NAD. Is this reaction more important under aerobic or anaerobic conditions? How are its isozymes used clinically? What is the metabolic fate of lactate? Among the metabolic problems seen in alcoholism are lacticacidosis and hypoglycemia. How does alcohol generate excessive NADH? Why would excessive NADH retard gluconeogenesis and cause lacticacidosis? Copyright, L. W. Stillway Revised 2005 Guide to Flying 14 Proteins with the same enzymatic activities are known as A. isomers. B. isozymes. C. polypeptides. D. protamers. E. stereoisomers. Lactate dehydrogenase is but one example of isozymes (isoenzymes). It contains four subunits, and five isozymes are possible. The H-type predominates in the heart and the M-type in skeletal muscle. The five tetramers are: H4, H3M,H2M2, H1M3 and M4. In case of a cardiac infarction, the heart forms will predominate. Since these isozymes are easily separated and their relative abundances determined by electrophoresis, the isozyme pattern is used quite frequently in clinical situations. 8. Revisited Refer to reaction 8. First, this is the glycerol 3-phosphate shuttle, and it occurs only in skeletal muscle. The metabolic purpose of this shuttle is to regenerate cytosolic NAD by transferring the electrons from cytosolic NADH to coenzyme Q of the mitochondrion. First, NADH reduces DHAP to glycerol 3-phosphate, which is transferred to through porin of the outer mitochondrial membrane. Glycerol 3-phosphate binds to glycerol 3-phosphate:Q reductase, which is located on the outer surface of the inner mitochondrial membrane. The electrons of glycerol 3-phosphate are first transferred to the prosthetic group FAD to form FADH2, then to coenzyme Q (Q), forming QH2. 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.8 8.9 8.10 This whole process is known as what shuttle? Is the inner mitochondrial membrane permeable to NADH? If electrons are passed on to mitochondrial FAD to form FADH2, What is the net yield of high-energy phosphate from cytosolic NADH? Is this shuttle operative under aerobic or anaerobic conditions? Why? Supposing conditions are aerobic and glycolysis is operating at a high rate, What would one expect to observe with respect to the concentration of lactate? Glycerol-3-P is required in the synthesis of fat (triglyceride, triacylglycerol). If carbohydrate is being rapidly converted to fat in adipose tissue, What effect would this have on the rate of the glycerol-3-P shuttle? Cytosolic NADH A. may be transported through the mitochondrial membrane where it may be oxidized back to NAD by NADH dehydrogenase. B. may be used to directly reduce NADP to NADPH through a transhydrogenase. C. is normally higher in concentration than NAD. D. may bind to the outer side of the inner mitochondrial membrane and be oxidized back to NAD, resulting in the oxidative phosphorylation of 3ADP to 3ATP. E. cannot be transferred into the mitochondrial matrix and is usually reoxidized by dihydroxyacetone phosphate. Cytoplasmic NADH cannot be transported into mitochondria. Under anaerobic conditions, it is usually reoxidized by LDH by reducing pyruvate to lactate, but this is a dead-end and limited because of the eventual accumulation of lactate. Under aerobic conditions, NADH can be oxidized by reducing DHAP to glycerol 3-P, which may be oxidized back to DHAP by a flavoprotein, glycerol 3-phosphate: reductase (an older term is glycerol 3-P dehydrogenase), located on the outer surface of the inner mitochondrial membrane. Thus, while NADH itself is not transferred into mitochondria, its electrons are. This is known as the glycerol 3-phosphate shuttle. Copyright, L. W. Stillway Revised 2005 Guide to Flying 15 12. Lactate metabolism and the Cori cycle 12.1 12.2 12.3 12.4 12.5 Under heavy muscular exertion, or any other condition that will produce a lack of tissue oxygen, what major glycolytic product will be produced? To which organ will this be carried by blood? Is this compound a major gluconeogenic one? What will this do to the concentration of NADH in the liver? What will be the probable fate of NADH (assume that the liver is aerobic)? Outline the Cori cycle. All of the following are gluconeogenic EXCEPT which one? A. β-Hydroxybutyrate B. Alanine C. Lactate D. Pyruvate E. Glycerol Gluconeogenic (glucogenic) compounds will yield a net synthesis of glucose. As a general rule, they must not yield acetyl CoA. Both alanine and lactate may be converted to pyruvate, which may be converted to oxaloacetate instead of being converted to acetyl CoA. The OAA may then be used for gluconeogenesis. With respect to the ketone bodies, β-hydroxybutyrate is converted to acetoacetyl CoA that will be split to two units of acetyl CoA, so they are not gluconeogenic. 13. Alanine 13.1 13.2 13.3 13.4 13.5 13.6 13.7 Which amino acid is the major gluconeogenic one? How is it converted to pyruvate? Name the enzyme and the coenzyme. What is ALT or GPT? Why is ALT important clinically? Outline the glucose-alanine cycle (alanine cycle). What is its function? Which of the following is the major glucogenic amino acid? A. Phenylalanine B. Aspartic acid C. Alanine D. Methionine E. Serine Copyright, L. W. Stillway Revised 2005 Guide to Flying 16 Of the amino acids listed, aspartate, alanine, methionine and serine are glucogenic, because they will yield a net synthesis of glucose. Phenylalanine is both glucogenic and ketogenic, because it yields fumarate, which is gluconeogenic and acetoacetic acid, which is one of the ketone bodies. Aspartate can be transaminated to OAA, and it may form malate, which is gluconeogenic. Alanine is the major glucogenic amino acid, because it is a major player in the glucose-alanine cycle. In this role, pyruvate is converted to alanine in skeletal muscle by transaminating amino groups from other amino acids. (See the Cori Cycle and the Glucose-Alanine Cycle.) Alanine serves as a neutral transport form of ammonia, and it is taken up by the liver, where it is transaminated back to pyruvate. The amino group is transferred to α-ketoglutarate forming glutamate. Pyruvate undergoes glucogenesis to glucose, which is transported back to muscle where it can be converted again to pyruvate, and the cycle starts over again. Liver glutamate passes its amino group on to the urea cycle as ammonia (ammonium ion). Methionine is converted through a series of reactions to succinyl CoA, making it gluconeogenic. 14. The pyruvate dehydrogenase complex Pyruvate dehydrogenase (PDH), now called the Pyruvate dehydrogenase complex (PDC) This reaction is most important because it bridges the gap between glycolysis and the TCA cycle. 14.1 14.2 14.3 14.4 14.5 14.6 14.7 14.8 14.9 Are the enzymes of PDC mitochondrial or cytosolic? Write the overall reaction. Name all of the cofactors involved and put them in sequence Pyruvate dehydrogenase is severely affected in alcoholism. Explain how this comes about. Can you name two other reactions that are influenced by this same factor (coenzyme or vitamin)? How is this enzyme complex regulated? Under what physiological conditions will it be inhibited? Is the reaction reversible? Does acetyl CoA contain a high-energy bond? Can acetyl CoA, or anything yielding acetyl CoA, result in a net synthesis of glucose by reversal of reactions covered so far? Why? Mark statements that are true of pyruvate dehydrogenase. A. Contains all enzyme activities on one peptide chain B. Requires lipoic acid C. Is located in the cytoplasm D. Produces NADH E. Utilizes biotin F. Is an enzyme complex consisting of three separate enzymes G. May be inactivated by covalent modification (phosphorylation) H. Is activated by insulin through dephosphorylation I. Forms carbon dioxide and acetyl CoA J. Has an analogous reaction mechanism to that of α-ketoglutarate dehydrogenase of the TCA cycle K. Utilizes cofactors in the order: TPP  lipoate  FADH2  NADH Correct answers: B, D, F, G, H, I, J, K 15. Pyruvate carboxylase 15.1 This reaction requires a coenzyme. What is it and with what type(s) of reaction(s) is it usually associated? 15.2 Name the enzyme. Can you name two other enzymes that utilize the same mechanism? If not we will get to them. Copyright, L. W. Stillway Guide to Flying 17 Revised 2005 15.3 15.4 15.5 This reaction is important under three metabolic conditions. Explain how it is important in situations requiring an energy demand, the synthesis of fatty acids and gluconeogenesis. How is this enzyme regulated? (This is a most important mechanism; know this modifier without fail). Explain how the reaction would be stimulated by oxidation, an energy demand or excess fuel. Which of the following enzyme velocities would increase when malonyl CoA levels are low? A. Pyruvate carboxylase B. Pyruvate dehydrogenase C. Fatty acid synthase D. Acetyl CoA carboxylase E. Glucose 6-phosphate dehydrogenase Malonyl CoA is generated by acetyl CoA carboxylase when fatty acid biosynthesis is stimulated. Malonyl CoA is a negative modifier of carnitine acyl transferase I (CAT I). Also called carnitine palmitoyl transferase I (CPT I). When malonyl CoA is high, β-oxidation is retarded, and when low, β-oxidation is stimulated and an abundance of acetyl CoA is produced in the matrix of mitochondria. In liver, this will result in the production of ketone bodies (Can you name them?) and acetyl CoA will stimulated pyruvate carboxylase. This enzyme, in fact, has an absolute requirement for acetyl CoA. So, when β-oxidation of fatty acids is stimulated, so is gluconeogenesis, in part from the stimulation of OAA production from pyruvate. This same mechanism is used to supply more OAA to the TCA cycle for triacylglycerol synthesis and in times of energy demand. 16. Glucoenogenesis 16.1 Define the term gluconeogenesis. This one is easy, the suffix ‘neo’ means new, so gluconeogenesis is the synthesis of glucose from noncarbohydrate precursors. On the other-hand, glucogenesis is all-encompassing. It includes carbohydrate sources, such as glycogen, as well as amino acids. Two major sources of glucose are lactate and amino acids--particularly alanine. All amino acids, except leucine (remember this one), are potential sources of glucose. It is axiomatic that in animals, even-carbon fatty acids are not sources of glucose, because they yield acetyl CoA. Plants, on the other hand, can yield a net synthesis of glucose from fatty acids via the glyoxylate pathway in which the two decarboxylations of the TCA cycle are by-passed. 16.2 16.3 Can you state two reasons why acetyl CoA will not yield a net synthesis glucose? Trace the pathways to glucose from lactate, alanine and methionine, and be able to recite each, especially the key and regulatory enzymes. (You do not need to know all of the reactions involved in methionine metabolism.) Copyright, L. W. Stillway Revised 2005 Guide to Flying 18 For example, while you are looking at the ceiling, recite the order of intermediates starting with lactate and ending with glucose: • Lactate • Pyruvate • Oxaloacetate • Malate • Oxaloacetate • Phosphoenolpyruvate Enzymes: • • • • • • lactate dehydrogenase pyruvate carboxylase malate dehydrogenase transport out of mitochondrion malate dehydrogenase PEP carboxykinase (key enzyme) Enzymes peculiar to gluconeogenesis (note that these are not in expressed in skeletal muscle): • PEP carboxykinase • Fructose 1,6-bisphosphatase • Glucose 6-phosphatase Irreversible steps in glycolysis and their enzymes: • Glucose  glucose 6-P (glucokinase, hexokinase) • fructose 6-P  fructose 1,6-bisphosphate (PFK-1) • PEP  pyr (pyruvate kinase) Key steps in gluconeogenesis and their enzymes: • oxaloacetate  PEP (PEPCK, phosphoenolpyruvate carboxykinase) • fructose 1,6-bisphosphate  fructose 6-phosphate (fructose 1,6-bisphosphatase) • glucose 6-phosphate  glucose (glucose 6-phosphatase) 16.4 Which two glycolytic-glucogenic enzymes are covalently modified when [cAMP] increases in response to either glucagon or epinephrine? Note that in gluconeogenesis, glyceraldehyde 3-phosphate dehydrogenase requires NADH, which must be supplied by other steps, i.e.. lactate dehydrogenase and cytosolic malate dehydrogenase. More on gluconeogenesis will come later. Which one of the following amino acids is strictly ketogenic? A. Alanine B. Methionine C. Tyrosine D. Isoleucine E. Leucine Leucine and lysine are the only amino acids that are strictly ketogenic. All of the others are either strictly glucogenic or mixed ketogenic and glucogenic. Copyright, L. W. Stillway Revised 2005 Guide to Flying 19 TCA Cycle, a few general questions: 1. How many oxidations are in the cycle? 2. How many involve NAD and FAD? 3. Where are the TCA cycle enzymes located? 4. What is meant by a substrate-level phosphorylation and give three examples. One from the TCA cycle. 5. If both carbons of acetyl CoA are labeled, what percentage will be released as carbon dioxide after the third turn? 6. Compared to other metabolic pathways, how rapid is the flux of carbons through the TCA cycle? 7. Name five biosynthetic pathways that are served by TCA cycle intermediates. 8. How many ATP (high-energy phosphate bonds) are produced in one turn of the cycle via the electron transport system? 9. How many high-energy phosphate bonds would be produced, when pyruvate dehydrogenase is included? 17. Citrate synthase 17.1 17.2 17.3 In part, this reaction drives the TCA cycle with the large free energy change available in the thioester linkage of acetyl CoA. How is this reaction related to the synthesis of fatty acids and steroids? Name the next enzyme. This enzyme asymmetrically binds citric acid, which results in the carbons of the acetyl portion of acetyl CoA not being released in the first turn of the cycle. Explain. 18. Isocitrate dehydrogenase 18.1 18.2 18.3 18.4 This is thought to be the major regulatory enzyme in the TCA cycle. If this step were inhibited, what effect would this have on the concentration of citrate and acetyl CoA? How is this enzyme regulated? Could this influence the rate of fatty acid synthesis? How? 19. α-Ketoglutarate dehydrogenase 19.1 19.2 19.3 19.4 19.5 19.6 19.7 What type of reaction is represented by isocitrate  α-ketoglutarate and αketoglutarate  succinyl CoA? In one turn of the cycle, how many carbons are lost as C02? What does this have to do with the non-gluconeogenic character of acetyl CoA? Other than isocitrate, name another major source of α-Kg. What type of reaction is involved? What is the coenzyme? The conversion α-ketoglutarate → succinyl CoA has virtually the same mechanism as a reaction we previously considered. Name it. Hint: the cofactors are TPP (Name the vitamin component and the type of reaction in which it is usually involved.), lipoate, FAD and NAD. How many ATP are produced in the cycle from each of the following to OAA via the electron transport system? • α-Kg • Succinyl CoA • Malate? Explain how α-KG serves a synthetic role in the synthesis of an amino acid. 19.8 19.9 Copyright, L. W. Stillway Revised 2005 Guide to Flying 20 20. Succinyl CoA synthase 20.1 20.2 20.3 20.4 20.5 20.6 20.7 20.8 20.9 The succinyl portion of succinyl CoA contains how many carbons? Is it potentially gluconeogenic? Which amino acids can yield succinyl CoA in an anaplerotic reaction? Which vitamin coenzyme does the last reaction making succinyl CoA from these amino acids require? You should know by now that this reaction generates GTP in a substrate-level phosphorylation. Where is GTP be used in gluconeogenesis? Name the enzyme. Is succinyl CoA a high-energy compound? How is this related to the synthesis of GTP from GDP? Further, how is this related to making the TCA cycle favorable in one direction? 21. Succinate:Q reductase (Succinate dehydrogenase) 21.1 21.2 21.3 21.4 Draw the structures of succinate and fumarate. As a general rule, FAD is often involved in reactions of this type, where a carbon-carbon double bond is introduced. Can you name another similar reaction that uses FAD? How about 34? This enzyme complex is a part of the inner mitochondrial membrane. It also contains FeS. What are two names for FeS? Using the sequence of reactions shown below, which reaction most likely uses FAD? CH2 CH2 A. CH CH B. OH CH CH2 C. O C CH2 D. Copyright, L. W. Stillway Revised 2005 Guide to Flying 21 This same sequence is seen in both the TCA cycle and β-oxidation. In the TCA cycle, the top compound would be succinate. In β-oxidation, it would be a saturated portion of a fatty acyl CoA. FAD often participates in reactions where a carbon-carbon double bound is created as in A. In B, water is added across the double bond, and in C, NAD oxidizes the β-hydroxy compound to a β-keto compound. D. would represent the formation of citrate in the TCA cycle or the production of acetyl CoA in β-oxidation. Fatty acid synthesis uses the reverse of these general reactions, except that two reductions are required, and they both use NADPH. 22. Fumarate 22.1 22.2 Name this enzyme. What was added across the double bond? How is malate important in gluconeogenesis? 23. Malate 23.1. 23.2. 23.3. 23.4. Name this enzyme. Draw the structures and show the reaction. What took place? Mammalian fatty acid synthase is most interesting, because seven enzyme activities are contained in a single peptide chain. Can you name two other proteins with at least two enzyme activities on the same peptide chain? (polyfunctional peptides) 24. Oxaloacetate 24.1 24.2 Supposing no TCA intermediates are available, how would citrate be made from glucose? Is the mitochondrial membrane permeable to acetyl CoA? If not, how is acetyl CoA made available to the cytosol for fatty acid synthesis? 25. Cytoplasmic citrate 25.1. 25.2. Name this enzyme. What are some of the fates of OAA? (See vicinities of 16 and 44). If it goes to 16, could this be a source of mitochondrial OAA? Is the mitochondrial membrane permeable to OAA? What are the fates of the acetyl CoA produced in the mitochondrion? 26. Fatty acid synthesis I 26.1. 26.2. 26.3. 26.4. 26.5. A most important regulatory step, name the enzyme and its coenzyme. Where did we encounter this type of reaction earlier? What is the regulatory influence of citrate on this reaction? Is this reaction the rate-limiting step in fatty acid biosynthesis What is the regulatory influence of fatty acyl CoA on this reaction? 27. Fatty acid synthesis II 27.1. 27.2. 27.3. If both carbons of acetyl CoA are labeled with 14C and it primes fatty acid synthase, in which end of the fatty acid will the label appear if unlabeled malonyl CoA is used? (Answer: The methyl end) When one palmitate is synthesized, how many malonyl CoA and acetyl CoA molecules are used? How many NADPH? If the C02 used by acetyl CoA carboxylase is labeled with 14C, will this label appear in the fatty acid? (No, why?) Guide to Flying Copyright, L. W. Stillway Revised 2005 22 27.4. 27.5. 27.6. 27.7. 27.8. In fatty acid synthesis, the growing acyl chain is bound to what component that functions as a swinging arm? What vitamin does it contain and with what coenzyme does it share most of its structural features? The series of reactions to 28 is very similar to a series in the TCA cycle. What is it? How many reductions occur in fatty acid synthesis? What coenzyme(s) is/are used? Name a major source of this coenzyme. What enzymes and reactions produce it? When fatty acid synthesis is stimulated, what other pathway would be expected to be stimulated for the production of NADPH? All of the following enzymes require biotin except which one? A. Phosphoenolpyruvate carboxykinase (PEPCK) B. Acetyl CoA carboxylase C. Pyruvate carboxylase D. Propionyl CoA carboxylase PEPCK does not require biotin. A deficiency of pantothenic acid would most likely impair the synthesis of A. biotin. B. 4'-phosphopantetheine. C. FAD. D. coenzyme A. E. palmitic acid. The vitamin pantothenic acid is a constituent of Coenzyme A and 4'-phosphopantetheine, so synthesis of these coenzymes would be impaired from a lack of panthothenate. Likewise, enzyme reactions, that utilize these coenzymes, such as the synthesis of palmitate, would be impaired. 28. Fatty acid synthesis III 28.1. 28.2. 28.3. Is palmitate the major fatty acid produced by fatty acid synthase? How are longer and shorter chains made? How are double bonds introduced? The reaction shown below requires which ones? (There is more than one answer.) C O O C SCoA SCoA A. B. C. D. E. F. a mixed-function oxidase NADPH NAD mitochondria cyt b5 O2 Copyright, L. W. Stillway Revised 2005 Guide to Flying 23 This represents the desaturation of stearic (as the CoA derivative) to oleic acid. Using shorthand notation it may also be written 18:0 Æ 18:1w9. Desaturation of fatty acids requires cyt b5, a mixedfunction oxidase. This enzyme is one of the many components of the cytochrome P450 system. In this case, there are four electrons transferred, two from the fatty acid and two from the co-reductant, NADPH. The products are oleic acid and two molecules of water, one of which contains the electrons from the fatty acid and the other contains electrons from the NADPH. Recall that the cytochromes contain iron that is part of a porphyrin ring. 29. Complex lipid synthesis 29.1. 29.2. 29.3. 29.4. Space did not permit the inclusion of the mechanisms for the synthesis of complex lipids, but you should be able to answer the following. How is the glycerol portion of the acylglycerols provided metabolically? Trace the production of this component from glucose. Can you recognize the distinguishing structural features of these? Phosphatidyl choline (lecithin) Phosphatidyl ethanolamine Phosphatidyl serine Ceramide Cerebroside Ganglioside Can you state the steps involved in the synthesis of these components? (The sequence is PS Æ PE Æ PC)? Which amino acid donates methyl groups to PE to form PC? In what form is the amino acid? 29.5. 29.6 The compound shown below NH2 COO H C CH2 CH2 + H3C S + NH3 N N N N CH2 H H O H H OH OH A. B. C. D. is homocysteine. is a precursor to phosphatidyl choline is found in high levels in cystathioninuria. is required for the synthesis of norepinephrine. The compound is S-adenosylmethionine, an activated form of methionine. Because of the high methyl group transfer potential, it is used metabolically to methylate compounds. For example, phosphatidyl ethanolamine can accept three methyl groups to form phosphatidyl choline. Epinephrine is synthesized from the methylation of norepinephrine, as shown below. The enzymes are called methyl transferases or transmethylases. Copyright, L. W. Stillway Revised 2005 Guide to Flying 24 HO H HO C HO + NH3 C H H SAM HO HO H C HO + H2N CH3 C H H 30. Cholesterol Transport 30.1 30.2 30.3 30.4 30.5 30.6 30.7 30.8 30.9 30.10 What are the main functions of VLDL? Where is it made? Where are chylomicrons produced? What do they mainly transport? Rank the following in order of density (lightest first): VLDL, LDL, chylomicrons and HDL. Name the enzyme that hydrolyzes triacylglycerols in VLDL and chylomicrons. How is it affected by insulin? Where is this enzyme found? What is the effect of heparin on the activity of this enzyme? What is the pathway below called? LDL Æ LDL receptor Æ lysosome Æ hydrolysis Æ free cholesterol Explain how free cholesterol regulates the affects listed. • • • 30.11 30.12 30.13 inhibition of cholesterol synthesis inhibition of receptor synthesis stimulation of ACAT The absence of LDL receptors results in what disease? Why? Is this pathway important in the regulation of cholesterol biosynthesis in extrahepatic tissues? Which one causes Familial hypercholesterolemia? A. Defective lipoprotein lipase B. Defective ACAT C. Defective LCAT D. Defective LDL receptors E. A diet high in cholesterol Familial hypercholesterolemia is caused by defective or absent LDL receptors. These are necessary to bind LDL so that the core of cholesteryl esters CE can be transported into the cell where the CE is hydrolyzed to free cholesterol and free fatty acid. This free cholesterol regulates the biosynthesis of HMG CoA reductase, the enzyme that catalyzes the main regulatory step in cholesterogenesis. It also regulates the production of LDL receptors and ACAT (Acyl CoA:cholesterol acyltransferase). ACAT esterifies free cholesterol by transferring a fatty acyl group from fatty acyl CoA Cholesterol + FACoA  CE + CoASH. Copyright, L. W. Stillway Revised 2005 Guide to Flying 25 Which of the following is the major source of unsaturated fatty acids in cholesteryl esters made in HDL with the catalysis of PCAT? A. Blood free fatty acids B. Blood triacylglycerols C. Phosphatidyl choline D. Fatty acyl CoA E. VLDL lipids PCAT is otherwise known as phosphatidyl choline:cholesterol acyltransferase. (The older name is LCAT for lecithin:cholesterol acyl transferase.) This enzyme, in association with HDL transfers a fatty acid from the number 2 position of phosphatidyl choline (lecithin) to free cholesterol, synthesizing a cholesteryl ester. Fatty acids occupying the number 2 position of phosphatidyl choline are usually unsaturated, such as linoleic, arachidonic, etc. This is in contrast to the number 1 position, which is usually saturated. The transfer is really quite simple. An acyl group is transferred from one hydroxyl to another. Products are lysophosphatidyl choline (phosphatidyl choline minus one fatty acid) and cholesteryl ester. A related reaction: The synthesis of prostaglandins and related compounds begins with which of the following? A. Hydrolysis of phosphatidyl choline by phospholipase A2 B. Cyclization of arachidonic acid by cyclooxygenase C. Release of free fatty acids from fat cells D. Stimulation of phospholipase A2 by steroids such as cortisol E. Inhibition of cyclooxygenase This is not involved with cholesterol transport but is important in wound healing and the inflammatory process. When an injury occurs, inflammation follows almost immediately. This is due to the action of certain compounds generated in the arachidonic acid cascade. The first step is the committed step. Arachidonic acid is hydrolyzed from the number 2 position of phosphatidyl choline by the action of phospholipase A2. In fact, is designated A2, because it is specific for fatty acids in this particular position of phosphatidyl choline. Phospholipase B, C and D do other things to the PC molecule. Steroids, such as cortisol, inhibit phospholipase A2. That is why they are known as antiinflammatory drugs. Cyclization of arachidonate by cyclooxygenase is an important step in that it is inhibited by non-steroidal anti-inflammatory drugs (NSAIDS), such as aspirin and ibuprofen. Note: Transport of lipids in water-soluble blood Free fatty acids (FFA) produced as a result of the hydrolysis of lipids (lipolysis in fat cells) are released into blood and are carried by albumin. Likewise, cholesterol triacylglycerols are transported by VLDL from the liver. Copyright, L. W. Stillway Revised 2005 Guide to Flying 26 People who have been fasting for some time have a decreased ability to transport free fatty acids in blood. This is because A. the liver produces less VLDL. B. albumin levels are low from conversion to amino acids and glucose. C. LDL levels are high enough to bind and not release most fatty acids. D. ketogenesis is fully active, so most energy from fat stores is in the form of ketone bodies. E. edema has damaged cell membranes so that free fatty acids freely diffuse throughout the body. One of the first physiological responses to starvation is breakdown of albumin as a source of amino acids, which can be converted to glucose in the liver. Albumin levels may therefore be low in starvation. This not only diminishes the ability to transport free fatty acids, but also osmotic pressure, and edema may result. 31. Lipolysis and lipogenesis TG DG FFA + GLYCEROL Pi PA BLOOD FACoA DHAP G-3-P You may find the above diagram helpful in your understanding of lipolysis and lipogenesis. 31.1 How could the availability of glycerol 3-P determine the rate of synthesis of triglycerides (triacylglycerols)? 31.2 In the fat cell, can insulin influence the availability of glycerol 3-P? 31.3 What metabolic effect does insulin have on lipolysis? 31.4 When lipolysis occurs, what are the products and what is the name of the enzyme? 31.5 What is the mechanism by which this enzyme is regulated by epinephrine, glucagon and other hormones? Copyright, L. W. Stillway Revised 2005 Guide to Flying 27 33. Chylomicrons, the exogenous pathway 33.1 To be metabolized, FFA must be "activated". This is accomplished by esterifying them to coenzyme A. The key to understanding how β-oxidation of fatty acids took place was the discovery of the transport role of carnitine. Without it, fatty acids will not enter mitochondria where enzymes for βoxidation are located. 33.2 33.3 33.4 Supposing carnitine is available, what two other components are required to transport fatty acids into mitochondria? Which is the main regulated component, and by what is it regulated? How is this regulation related to fatty acid biosynthesis and ketogenesis? A carnitine deficiency (lack of the ability to synthesize sufficient carnitine) would most likely result in which one of the following? A. Diminished ketogenesis B. Elevated blood free fatty acids C. Lack of fatty acyl CoA in the mitochondrial matrix D. Increased utilization of liver glycogen reserves E. All of the above. Some individuals do lack carnitine. Since this compound is needed to transport fatty acids into the matrix of mitochondria, a deficiency of it would result in a lack of fatty acid supply to the mitochondrial β-oxidation scheme. Less acetyl CoA would be generated from fatty acids, and the individual would be more dependent on carbohydrates as a source of energy. Ketogenesis would be decreased because of the lack of acetyl CoA. In a person who consumes a high protein diet all of the following are most likely except which one? A. Elevated ketogenesis B. Increased levels of PEPCK C. Decreased CAT I activity D. Increased levels of mitochondrial acetyl CoA E. Decreased transport of citrate out of mitochondria A high protein diet is low in carbohydrate. Carnitine acyl transferase I activity would increase rather than decrease, because When carbohydrates are lacking, β-oxidation, ketogenesis and glucogenesis are turned on, and fat synthesis is turned off. Acetyl CoA is produced in greater amounts from fatty acids, because the inhibition of CAT I has been removed, since very little malonyl CoA is available due to the lack of carbohydrate. Ketogenesis is elevated. PEPCK activity is elevated because of the increased gluconeogenesis, since dietary carbohydrate is lacking. Less citrate is transported out of mitochondria, because the synthesis of fat is diminished. 34. Beta-Oxidation of fatty acids 34.1 This sequence of reactions is the reverse of that involved in the biosynthesis of fatty acids. 24.2 How does it differ? (location, cofactors). Recall the same series in the TCA cycle. 34.3 What is/are the product(s) of β-oxidation from even carbon fatty acids? Odd-carbon fatty acids? 34.4 What are the cofactors involved in β-oxidation? Which of the following is required in fatty acid biosynthesis, as opposed to β-oxidation of fatty acids? Copyright, L. W. Stillway Revised 2005 Guide to Flying 28 A. B. C. D. E. NADH FAD NADP CoASH NADPH Fatty acid biosynthesis requires fatty acid synthase. The fatty acyl chain is covalently linked to ACP (acyl carrier protein portion of mammalian FAS and an individual protein in plants and bacteria). The two reductive steps require NADPH as a source of electrons. In β-oxidation, the fatty acyl chain is covalently linked to coenzyme A, and it is oxidized first by FAD and then NAD. E is the correct answer. 35. Regulation by acetyl CoA levels 35.1 Suppose energy demand is not high, the TCA cycle is shut down, and that acetyl CoA accumulates, what are the metabolic consequences? For example: (l) How would this stimulate gluconeogenesis? (2) How would fatty acid synthesis be stimulated? (3) How would β-oxidation be inhibited? 35.2 On the other hand, suppose that there is an energy demand, how would the level of acetyl CoA stimulate the production of ATP? Assume that ATP and NADH are low. All of the following are inhibited by ATP except which one? A. phosphofructokinase B. citrate synthase C. isocitrate dehydrogenase D. pyruvate dehydrogenase E. hexokinase All of the above enzymes, except hexokinase, are inhibited by ATP. When ATP levels decrease, these enzyme activities will increase. At elevated concentrations, which of the following will decrease the activity of pyruvate dehydrogenase? A. Acetyl CoA B. Coenzyme A C. NAD D. ADP E. Ca2+ Acetyl CoA is the only component listed that will decrease the activity of PDH. Actually, it is high ratios of acetyl CoA/coenzyme A, NADH/NAD, ATP/ADP that decrease PDH activity. Calcium increases PDH activity by stimulating dephosphorylation, as does insulin. So, when the phosphatidyl inositol pathway is stimulated, one of the results will be stimulation of PDH. 36. Thiokinase and HMG CoA Synthase 36.1 Ketogenesis is associated with diabetes, fasting and low carbohydrate diets, eg, those high in protein or fat. Generally, when fat stores are mobilized, ketogenesis will follow. This Copyright, L. W. Stillway Revised 2005 Guide to Flying 29 occurs when carbohydrates are unavailable. Perhaps a better way of looking at it is the level of acetyl CoA. If it is excessive, much of it will be shunted into the synthesis of ketone bodies. Can you name the three ketone bodies? 36.2 The first step in ketogenesis involves the condensation of two acetyl CoA molecules to form acetoacetyl CoA. Is this the same reaction that occurs in cholesterogenesis? Is it the reverse of the last step in β-oxidation? 36.3 Hydroxymethylglutaryl CoA (HMGCoA) is a common intermediate in both ketogenesis and cholesterogenesis. Obviously, if it is synthesized in the liver mitochondrion, it forms ketone bodies, but if formed in the cytoplasm, it is made into cholesterol. This is a good example of separate metabolic pools to define different pathways using the same intermediate. 36.4 What tissue is the major producer of cholesterol, ketone bodies and glucose? All of the following are known as ketone bodies (or ketones) except which one? A. α-ketobutyrate B. β-hydroxybutyrate C. Acetoacetic acid D. Acetone α-ketobutyrate is not one of the ketone bodies. Which of the following enzymes is used exclusively in ketogenesis? A. HMG CoA synthase B. HMG CoA reductase C. HMG CoA lyase D. β-hydroxybutyrate dehydrogenase E. 3-ketothiolase In ketogenesis, two units of acetyl CoA are condensed by thiolase to produce acetoacetyl CoA. This is the reversal of the last step of β-oxidation. A third acetyl CoA unit is added by the catalytic action of HMG CoA synthase. All of these steps are also found in cholesterogenesis. In ketogenesis, HMG CoA lyase cleaves HMG CoA to acetoacetic acid and acetyl CoA. This is the only enzyme listed that is unique to ketogenesis. In cholesterogenesis, HMG CoA is reduced to mevalonic acid by HMG CoA reductase using 2NADPH, the committed step. β-Hydroxybutyrate dehydrogenase is found in the inner membrane of mitochondria, and it reduces acetoacetic acid to β-hydroxybutyric acid or vice versa. Why are ketone bodies made only in the liver and kidney? The secret is HMG CoA synthase. This enzyme is not present in the mitochondria of other tissues. It is present in the cytoplasm, however, where it is used in the synthesis of cholesterol. Interestingly, the HMG CoA lyase is present in the mitochondria of cells other than those of the liver and kidney, because there are other small sources of HMG CoA, such as in the catabolism of leucine. They therefore can make small amounts of ketone bodies, but overall, the liver is the major producer and other tissues are consumers. 37. Ketogenesis II 37.1 Name this enzyme. HMG CoA lyase (lyases split molecules) produces acetoacetate from HMG CoA. 37.2 37.3 37.4 Can you draw or recognize the structure of HMG CoA? Is HMG CoA lyase found in places other than liver mitochondria? If so, where? Guide to Flying Copyright, L. W. Stillway Revised 2005 30 38. Ketogenesis III 38.1 38.2 38.3 38.4 38.5 The conversion, acetoacetate  β-hydroxybutyrate occurs in the inner mitochondrial membrane. The enzyme is β-hydroxybutyrate dehydrogenase. What is the coenzyme, and what determines the ratio of acetoacetate/β-hydroxybutyrate? In muscle or adipose tissue cells, the above enzyme is also present in the inner mitochondrial membrane. What purpose does this serve? (It has to do with the utilization of ketone bodies.) The utilization of acetoacetate in extrahepatic tissues requires a TCA-cycle intermediate to activate acetoacetate. Name the compound that donates the CoA. How is acetoacetyl CoA degraded in extrahepatic cells? 39. Ketogenesis IV 39.1 39.2 39.3 39.4 Do the ketone bodies normally circulate in blood? Are they water-soluble? How is acetone produced? (ie, does it require an enzyme?) Are they ever found in urine? When? (two circumstances) 40. Cholesterogenesis 40.1 40.2 You should know by now that ketogenesis occurs in liver mitochondria and that cholesterogenesis occurs in the cytoplasm. Can HMG CoA cross the mitochondrial membrane? 41. Cholesterogenesis regulation 41.1 41.2 41.3 41.4 41.5 Is this an important regulatory step in cholesterogenesis? What is the name of the enzyme? What is the main mechanism by which this enzyme is regulated? What does this step have to do with the LDL pathway? Name a few drugs that specifically inhibit this enzyme. 42. From mevalonate to cholesterol 42.1 42.2 42.3 42.4 42.5 Is squalene an intermediate in cholesterol biosynthesis? Vitamin D3 is made by exposure of 7-dehydrocholesterol, which is made in the cholesterol synthetic pathway. Isoprenoids are also made from cholesterol synthetic intermediates, including coenzyme Q (ubiquinone) and dolichol. What does the literature say about the administration of HMG CoA synthase inhibitors (statin drugs) and the supply of such compounds as coenzyme Q and dolichol? Severe muscle pain is experienced by some people who take statin drugs. How could this be metabolically explained? All of the following are intermediates in cholesterogenesis except which one? A. Geranyl pyrophosphate B. Squalene C. Lanosterol D. HMG CoA E. Glycocholic acid Glycocholic acid is one of the two main bile acids. Taurocholate is the other major bile acid Copyright, L. W. Stillway Revised 2005 Guide to Flying 31 The enzyme HMG CoA lyase is found mostly in which of the following tissues or cellular compartments? A. Liver mitochondria B. Skeletal muscle cytoplasm C. Surface of endothelial cells D. Kidney cytoplasm E. Brain cytoplasm HMG CoA lyase splits HMG CoA to acetoacetate and acetyl CoA, and it is located mainly in liver mitochondria. 43. Fates of cholesterol 43.1 43.2 43.3 43.4 43.5 43.6 43.7 43.8 43.9 43.10 43.11 43.12 43.13 43.14 43.15 43.16 43.17 43.18 43.19 43.20 43.21 43.22 What major type of reaction is utilized to produce bile acids from cholesterol? In which organ does this occur? What types of enzymes carry out this kind of reaction? Are they important in the detoxication or metabolism of drugs? What is a conjugated bile acid? What happens if bile acids cannot be conjugated? What group(s) is/are conjugated with bile acids? How is cholesterol metabolized, ie, broken down and excreted? Cytochrome p450 catalyzes what main type of reaction? Check the reaction above 43. There are at least two enzymes that will catalyze the esterification of cholesterol to cholesteryl esters. Name these two enzymes. (Abbreviations are OK.) Where are these two enzymes found? How is cholesterol stored? How is it transported from the liver? How is it transported from tissues to the liver? What is the main result of very low levels of HDL? Where are chylomicrons made? Where are they first degraded? What enzyme catalyzes the degradation of chylomicrons and VLDL? Where is it located? With respect to this enzyme, what is the main pathological result if it is deficient? What does the term "clearing" mean with respect to chylomicrons? Which of the following is the most likely explanation for a constantly high VLDL level? A. Familial hypercholesterolemia B. Defective LCAT C. Defective lipoprotein lipase D. Lack of LDL receptors E. Necrotic liver Lipoprotein lipase is found on the surface of capillary endothelial cells, and it is required to hydrolyze triacylglycerols in VLDL and chylomicrons. If defective or lacking, VLDL and/or chylomicrons could be high. 44. Transamination (transaminases or amino transferases) See at bottommiddle of map. 44.1 44.2 44.3 What type of reaction is usually involved in the first step of amino acid degradation? Name the coenzyme. Which α-keto acid usually participates in this reaction? Guide to Flying Copyright, L. W. Stillway Revised 2005 32 44.4 44.5 44.6 44.7 44.8 Why would amino groups from the 20 different amino acids be transferred primarily to only one α-keto acid to produce glutamate? What are two other major transaminases that are routinely measured? Where do they fit into the metabolic scheme? Which ones are determined clinically? In what organ are most amino acids degraded? Which of the following is a coenzyme required for transamination reactions? A. Biotin B. Pyridoxal phosphate C. NAD D. NADP E. Carnitine Transamination reactions require the coenzyme pyridoxal phosphate. 45. Urea cycle 45.1 Take a look at the urea cycle. 45.2 Where do the two nitrogens in urea originate? (The classic answer is ammonia and aspartate). 45.3 What common amino acid supplies both? 45.4 Where is the urea cycle located in the body? 45.5 What if a patient has a urea cycle defect or has severe liver damage, what kind of diet would be appropriate so as to minimize hyperammonemia? Defective ornithine transcarbamoylase will most likely result in which of the following? A. Accumulation of arginine B. Hyperammonemia C. Accumulation of citrulline D. Lack of carbamoyl phosphate E. Accumulation of argininosuccinate Ornithine transcarbamoylase (OTCase) catalyzes the condensation of ornithine and carbamoyl phosphate in the urea cycle Hyperammonemia is a condition that is produced when the urea cycle is impaired, such as in alcoholism, poisoning by chlorinated hydrocarbons, hepatitis and urea cycle enzyme defects, such as OTCase. (Another OTCase is involved in pyrimidine biosynthesis.) 46. Glutamate dehydrogenase & Glutaminase 46.1 Two enzymes in this vicinity produce ammonia, what are they? 46.2 What is the major function of glutamine here? 46.3 Why would these reactions produce ammonia? 47. Creatine phosphate 47.1 47.2 47.3 47.4 47.5 47.6 47.7 What is CP? Is ATP required in its synthesis? Is CP produced in the cytosol? For what purpose? Name this enzyme. How does it differ from carbamoyl phosphate synthase in the cytosol? How do they differ in name? Guide to Flying Copyright, L. W. Stillway Revised 2005 33 48. Ornithine 48.1 48.2 48.3 48.4 Is ornithine found in proteins? Is it an amino acid? What type? If ornithine is decarboxylated by ornithine decarboxylase to produce carbon dioxide, what is the other major product? 49. Aspects of the urea cycle 49.1 49.2 49.3 49.4 What are the products of the urea cycle? Which product is also a TCA cycle intermediate? Can you recognize urea cycle enzyme names and differentiate them from other enzymes? Fumarate will not cross the mitochondrial membrane. If it can be converted to malate, how might it be metabolized? See how it picks up an amino group from glutamate? This enzyme is aspartate transaminase (AST, aspartate aminotransferase), and it is a major liver enzyme. In fact, AST and ALT concentrations in blood are used to diagnose liver problems (liver function test). 50. Arginine 50.1 50.2 50.3 50.4 50.5 50.6 50.7 50.8 50.9 50.10 50.11 Is arginine an amino acid? Is it essential? Is it basic or acidic? Is it a constituent of proteins? What purpose does it serve in the urea cycle? Name this enzyme. In which organ is urea made? Where is it excreted? Assume that insulin is low or lacking, as in IDDM. What effect does this have on proteolysis, blood amino acids and urea output? What is the metabolic fate of most amino acid carbon skeletons? (This concept is important.) 51. Met, Ile, and Val (lower left of map) 51.1 51.2 This pathway is important, because it requires the coenzyme form of vitamin Bl2 or cobalamin. The important reactions for our purposes are 53 & 54. What is the name of the disorder in which Bl2 is deficient in the body but not in the diet? 52. Production of propionyl CoA 52.1 How is this reaction similar to pyruvate dehydrogenase and α-Kg dehydrogenase? 53. Metabolism of propionyl CoA 53.1 53.2 53.3 What are the main metabolic sources of propionyl CoA? (Name the compounds.) Do odd-carbon fatty acids furnish a significant amount of propionyl CoA? Is propionyl CoA gluconeogenic? Copyright, L. W. Stillway Revised 2005 Guide to Flying 34 54. Methylmalonyl CoA 54.1 54.2 54.3 54.4 54.5 54.6 Name a reaction or enzyme in which the coenzyme form of Bl2 or cobalamin is used. If a Bl2 deficiency exists, what will accumulate (more than one compound is possible) and be excreted in the urine? Could the person be acidotic? What protein secreted by the stomach is lacking in pernicious anemia? What type of protein is it? Why is it necessary for Bl2 metabolism? Cobalamin is also involved in a reaction having to do with nucleotides. What is the reaction? How is this reaction related to pernicious anemia? 55. Electron transport & oxphos I 55.1 55.2 55.3 55.4 55.5 55.6 55.7 55.8 55.9 55.10 55.11 Where are NADH and FADH2 located for the purpose of oxidative phosphorylation, ie, do they cross the mitochondrial membrane system? What is the driving force for oxidative phosphorylation? What is the main thesis of the chemiosmotic hypothesis? How many high-energy phosphate bonds actually result metabolically from the oxidation of one FADH2 and one NADH? How is respiratory control defined? In what mitochondrial compartment does electron transport and oxidative phosphorylation occur? What is the main function of the electron transport system? Name the enzyme that synthesizes ATP in mitochondria. Is 2,4-dinitrophenol (DNP) an inhibitor or uncoupler of the electron transport system? By what mechanism does it act? Which protein does oligomycin inhibit? How would one go about differentiating between oligomycin and an inhibitor of the electron transport system? Which of the following is NOT a property or component of the mitochondrial electron transport system A. Proton pumping B. Cytochrome c C. Ubiquinone D. F oF 1ATPase E. Cytochrome oxidase F oF 1ATPase is an enzyme associated with the phosphorylation of ADP. Like the electron transport system, it is located in the inner membrane of mitochondria, but it is not a part of the electron transport system. For example, it can be uncoupled from the electron transport system with dinitrophenol. 56. Electron transport & oxphos II 56.1 56.2 56.3 56.4 56.5 56.6 What is a flavoprotein? Name the vitamin component? What is another name for Co Q? Describe a cytochrome, ie, what are its two components? What is the order of cytochromes in terms of electron transfer? How many electrons are transferred by a cytochrome? Differentiate between an inhibitor of electron transport and an uncoupler of phosphorylation. Guide to Flying Copyright, L. W. Stillway Revised 2005 35 56.7 . Which of the following is/are uncouplers or inhibitors? Cyanide Oligomycin Rotenone Amytal Dinitrophenol 56.8 56.9 What are the P:O ratios for succinate and malate? Will oxidative phosphorylation occur with the combination of succinate and rotenone? What will be the P:O ratio? 57. Electron transport & oxphos III 57.1 57.2 57.3 57.4 57.5 57.6 Of what significance is a pH gradient across the inner mitochondrial membrane? In living cells, is the pH higher or lower on the outside than the inside of a mitochondrion? Is a voltage generated across the membrane? What is the origin of protons on the outside of the membrane? Is the energy for phosphorylation of ADP provided by a free energy difference or electron spin? How many sites in the electron transport system provide enough free energy change for the phosphorylation of ADP? How many high-energy phosphate bonds can result from this reaction? (58 is to be found as part of the glycerol 3-P shuttle.) 58. Electron transport & oxphos IV 58.1. 59. Electron transport & oxphos V 59.1 59.2 59.3 These projections on the inner membrane contain the ATPase that phosphorylates ADP. (It is called an ATPase, because it will also hydrolyze ATP to ADP and Pi.) Where is the influx of protons controlled? Explain how dinitrophenol can uncouple mitochondria while phosphorylation is inhibited by oligomycin. The protein effected by oligomycin is designated by what abbreviation 60. Electron transport & oxphosphorylation VI 60.1 60.2 60.3 60.4 What is another name for cyt a + a3? Is this cytochrome inhibited by cyanide and carbon monoxide? What blood component is also affected by cyanide and carbon monoxide? If 20 umoles of O2 are consumed in the process of synthesizing 10 mmoles ATP, what is the P:O ratio? Ans. 0.25 Cytochrome c is known for all of the following except which one? A. Water solubility B. Large apoprotein C. Contains heme D. Species homology E. Accepts only one electron at a time Copyright, L. W. Stillway Revised 2005 Guide to Flying 36 Cytochrome c consists of a protein (apoprotein) and heme. The protein component is relatively small when compared to the other mitochondrial cytochrome proteins, and it has a high degree of homology in its primary structure across a wide variety of species. Cytochrome c, like most of the other cytochromes accepts only one electron at a time. The heme iron reversibly accepts the electron. The terminal electron acceptor in the mitochondrial electron transport system is A. Cytochrome oxidase. B. Coenzyme Q10. C. Cytochrome p450. D. Cyclooxygenase. E. Cytochrome c. The final electron acceptor is cytochrome oxidase (cytochrome a + a3), which may be inhibited by cyanide, carbon monoxide, etc. It transfers four electrons to molecular oxygen O2 forming water. 61. 62. 63. 64. Electron transport & oxphosphorylation VI Mitochondrial phosphate carrier ATP-ADP translocase Porphyrin metabolism Copyright, L. W. Stillway Revised 2005 Guide to Flying 37 Question and Answer Exercise Section Predict what metabolic intermediates will accumulate if the following enzymes are genetically lacking or defective. Also predict which products will be short in supply. Defective Enzyme Accumulates Short in Supply 3-P glycerate, ATP 3-P glycerate kinase 1,3-bisphosphoglycerate, ADP Carnitine acyl transferase Hexosaminidase A Acetyl CoA carboxylase HMG CoA reductase β-Hydroxybutyrate dehydrogenase Fructose bisphosphatase Glucose 6-phosphatase Argininosuccinase GPT Carnitine synthase Lipoprotein lipase GOT Ornithine transcarbamoylase Glutamate dehydrogenase Phosphoglucomutase Pyruvate kinase Fatty acid synthase Succinate dehydrogenase Phosphorylase pyruvate carboxylase Isocitrate dehydrogenase Citrate lyase Pyruvate dehydrogenase α-Ketoacid dehydrogenase Phosphofructokinase Copyright, L. W. Stillway Revised 2005 Guide to Flying 38 Regulation of Enzyme Activity. Which of the following enzymes are regulated by phosphorylation catalyzed by a protein kinase? If so, is the activity increased or decreased? 1. 2. 3. 4. 5. 6. 7. 8. Malate dehydrogenase Isocitrate dehydrogenase Phosphofructokinase Glycogen synthase Hormone sensitive lipase Pyruvate dehydrogenase Phosphorylase Phosphorylase kinase Answers: 1. No 2. No 3. No, but PFK-2 is inactivated when phosphorylated. 4. Yes, less active, active in presence of G-6-P. 5. Yes, more active (lipolysis is stimulated) 6. Yes, less active 7. Yes, it is phosphorylated by phosphorylase kinase, which is activated by a cAMP-dependent protein kinase. 8. Yes, more active (activates phosphorylase) Copyright, L. W. Stillway Revised 2005 Guide to Flying 39 Which of the following are directly regulated by energy charge? 1. Phosphorylase b 2. Isocitrate dehydrogenase 3. Pyruvate dehydrogenase 4. Phosphofructokinase 5. Glucose 6-phosphatase 6. Fructose 1,6-bisphosphatase 7. Citrate synthase 8. NADH dehydrogenase 9. Lactate dehydrogenase 10. Glutamate dehydrogenase 11. Phosphorylase a 12. PFK-2 Answers: 1. Activated by AMP (allosteric): Phosphorylase b is normally the inactive form of this enzyme, but at high[AMP], it is allosterically stimulated. 2. Activated by AMP, inhibited by high ATP and inhibited by NADH (allosteric) 3. Inactivated by high ATP and acetyl CoA, which result in phosphorylation of one of the enzyme proteins of the complex (covalent modification) 4. Inhibited by high ATP and citrate and stimulated by high AMP. (ATP is bound to two different sites: one catalytic, the other allosteric). Fructose 2,6-bisphosphate stimulates. 5. Not regulated allosterically. 6. Inhibited by AMP, stimulated by ATP and citrate. Fructose 2,6-bisphosphate inhibits. 7. Inhibited by ATP. 8. Not known to be regulated directly. This is the first enzyme in the mitochondrial electron transport system. 9. Not known to be directly regulated. 10. GTP and NADH inhibit, ADP stimulates (A low energy charge will stimulate the production of α-Kg that can be used in the TCA cycle to stimulate energy production.) 11. Not directly regulated by energy charge. The primary regulation is accomplished by covalent modification in which the enzyme protein is phosphorylated by phosphorylase kinase in response to the cAMP cascade or to increased [Ca++], which activates calmodulin, one of the subunits of phosphorylase kinase. 12. Stimulated by fructose 6-P. PFK-2 activity is part of a tandem enzyme protein that also has fructose 2,6-bisphosphatase-2 (FBPASE-2) activity Copyright, L. W. Stillway Revised 2005 Guide to Flying 40 Match the impaired reaction or process with a vitamin or coenzyme deficiency: 1. Homocysteine  methionine 2. proline  hydroxyproline 3. pyruvate  acetyl CoA 4. isocitrate  α-ketoglutarate 5. alanine  pyruvate 6. methylmalonyl CoA  succinyl CoA 7. dUMP  dTMP 8. glutamate  α-ketoglutarate + NH3 9. HMGCoA  mevalonate 10. propionyl CoA  succinyl CoA 11. propionyl CoA  methylmalonyl CoA 12. lactate  pyruvate 13. 13-cis retinal  all trans retinal 14. R C R CH CH COOH O (stimulated) O H 15.Ca+2 gut  Ca+2 blood 16. conversion of prothrombin to active form . A. B. C. D. E. F. G. H. I. J. K. L. M. N. COOH thiamine pantothenic acid riboflavin folic acid cobalamin lipoic acid nicotinic acid pyridoxine ascorbate biotin vitamin A vitamin E vitamin D vitamin K CH Answers: 1. 2. 3. 4. 5. 6. 7. 8. E I A, B, C, F, G G H E D, E G 9. 10. 11. 12. 13. 14. 15. 16. G J, E J G K L M N Copyright, L. W. Stillway Revised 2005 Guide to Flying 41 Match the process with the compartment 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. glycerol 3- P  DHAP A. plasma membrane R - CH3  R-CH20H B. cytosol glucose  lactate C. ER acetyl CoA  malonate D. all mitochondria acetyl CoA  cholesterol E. outer mitochondrial membrane acetyl CoA + OAA  citrate F. inner mitochondrial membrane glycogen  g-l-P G. mitochondrial matrix palmitate  8 acetyl CoA 6 Pi + 6 ADP + 2 NADH + 2O2  6 ATP + 2NAD + 2H20 lactate  pyruvate acetyl CoA + 7 malonyl CoA + 14 NADPH  palmitate + 14 NADP + 8 CoA ATP  c-AMP Answers: 1. 3. 5. 7. 9. 11. B, F B B B, D F B 2. 4. 6. 8. 10. 12. C B G G B A Copyright, L. W. Stillway Revised 2005 Guide to Flying 42 Match the process or item with the compartment 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16 . 17. 18 . 19. 20. 21. 22. 23. 24. 25. 26. 27. glycolysis TCA cycle oxidative phosphorylation glycogen β-oxidation fatty acid synthesis mixed-function oxidases cyt b5 cyt p450 cyt b adenylate cyclase cAMP dependent protein kinase albumin FFA glucagon receptor insulin receptor phosphofructokinase I ketogenesis gluconeogenesis cholesterogenesis carnitine palmitoyl transferase I carnitine palmitoyl transferase II triacylglycerol synthesis steroid hormone receptors peptide hormone receptors thyroid hormone receptor retinoic acid receptor vitamin D3 receptor A. B. C. D. E. F. G. H. I. J. K. plasma membrane endoplasmic reticulum mitochondrial matrix cytoplasm blood inner mitochondrial membrane mitochondrial membrane outer mitochondrial. membrane liver mitochondria cytosol nucleus Answers: 1. 3. 5. 7. 9. 11. 13. 15. 17. 19. 21. 23. 25. 27. D and J F C D, B (ER is cytoplasmic) D, B (ER is cytoplasmic) A E A I D, B F D or J K K 2. 4. 6. 8. 10. 12. 14. 16. 18. 20. 22. 24. 26. C D or J D or J D, B (ER is cytoplasmic) F D or J A (muscle excluded) D or J (I) D or J (begins in liver mitochondria) F D, B (ER is cytoplasmic) A K Copyright, L. W. Stillway Revised 2005 Guide to Flying 43 Match the following processes with appropriate factors that should stimulate (More than one might be appropriate.): 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Answers: 1. 2. 3. 4. 5. B, C, E, H A C C, D B, C, E 6. 7. 8. 9. 10. F C, D G F B, E glycogen  G-l-P (liver) acetyl CoA  palmitate F-6-P  PEP pyruvate  acetyl CoA glycogen  G-1-P glucose  triglyceride Isocitrate  α-ketoglutarate pyruvate  oxaloacetate UDP glucose  glycogen triglyceride  glucose A. B. C. D. E. F. G. H. citrate glucagon AMP NAD epinephrine insulin acetyl CoA Pi Match the following processes with appropriate factors (one or more) that should inhibit or retard: 1. 2. 3. 4. 5. 6. 7. 8. 9. Answers: 1. 2. 3. 4. 5. C, D, F, H B, C A C, F D 6. 7. 8. 9. E, H, I G J J glucose  pyruvate pyruvate  acetyl CoA triglyceride  FFA + glycerol F-6-P  F-1,6-P glucose  glucose -6-P UDP glc  glycogen+1 acetyl CoA  malonyl CoA acetyl CoA  squalene HMGCoA  mevalonate A. B. C. D. E. F. G. H. I J. insulin acetyl CoA ATP G-6-P cAMP citrate palmitoyl CoA glucagon epinephrine cholesterol Copyright, L. W. Stillway Revised 2005 Guide to Flying 44 Match the process or observation on the left with the appropriate circumstance on the right. More than one circumstance may be correct. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. elevated glucagon elevated insulin depressed glucagon depressed insulin hyperglycemia hypoglycemia acetoacetate in urine glucosuria defective lysosomal hydrolase defective or absent LDL receptors ketosis (ketonemia) positive nitrogen balance negative nitrogen balance accelerated gluconeogenesis accelerated glycolysis hyperammonemia glycogenesis glycogenolysis lipogenesis lipolysis proteolysis, proteogenesis depressed ketoacid decarboxylase defective hydroxylase accumulated sphingomyelin GM2 accumulation severe atherosclerosis at age 14 A. B. C. D. E. F. G. H. I. J. K. L. M. N. O. P. fasting adolescent diabetes alcoholism during exercise post exercise resting after meal low carbohydrate diet high fat diet high protein diet low protein diet anoxia Niemann Pick disease PKU maple syrup urine disease familial hypercholesterolemia essential amino acid deficiency Answers: 1. 2. 3. 4. 5. 6. 7. A,C(possibly),D,G,H,I E,F,J E,F,J A,B,D,G,I B C A,B,H,I 8. 9. 10. 11. 12. 13. 14. B L O A,B,D,G,H,I F,I A,B,C,D,J A,B,D,F,G,H,I 15. 16. 17. 18. 19. 20. 21. D,K C,I,P(w proteolysis) E,F A,B,D,G E,F,H A,B,D,G,I A,B,D,H,J 22. 23. 24. 25. 26. N M L P O Copyright, L. W. Stillway Revised 2005 Guide to Flying 45 Name the coenzyme utilized in the following reactions: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. Aspartate + α-Kg  OAA + glutamate HMG CoA + 2NADPH  Mevalonate Acetoacetate  β-Hydroxybutyrate malate  oxaloacetate lactate  pyruvate glyceraldehyde 3-P  1,3-bisphosphoglycerate pyruvate  acetyl CoA succinate  fumarate methylmalonyl CoA  succinyl CoA alanine  pyruvate α-ketoglutarate  succinyl CoA acetyl CoA  malonyl CoA proline  hydroxyproline Answers: Pyridoxal phosphate NADPH NADH NAD NAD NAD (TPP, lipoate, FAD, NAD) FAD B 12(cobalamin) pyridoxal phosphate (TPP, lipoate, FAD, NAD) biotin ascorbate. Copyright, L. W. Stillway Revised 2005 Guide to Flying 46 Which of the following have analogous reaction mechanisms? Write the analogous reaction at the right. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. alanine −−> pyruvate pyruvate  acetyl CoA acetyl CoA  malonyl CoA OAA + acetyl CoA  citrate aspartate  oxaloacetate acetoacetyl CoA + acetyl CoA  HMGCoA pyruvate  oxaloacetate α-Kg  succ CoA propionyl CoA  methylmalonyl CoA glutamate  α-Kg Answers: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 5,10 or any transamination 8 (oxidative decarboxylation) 7, 9 carboxylation (requires biotin) 6 (condensation) 1, 10 (transamination) 4. (condensation) 3, 9 (carboxylation) 2 (oxidative decarboxylation) 3, 7 (carboxylation (requires biotin) 1, 5 (transamination or ox deamination with glu dehydrog.) Copyright, L. W. Stillway Revised 2005 Guide to Flying 47 For the following enzymes, write in the appropriate coenzyme(s) and the vitamin component: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. acetyl CoA carboxylase alanine transaminase isocitrate dehydrogenase succinate dehydrogenase pyruvate dehydrogenase fatty acid synthase pyruvate carboxylase methylmalonyl CoA mutase leucine transaminase carrier of acetyl groups carnitine acyl transferase I & II (carnitine palmitoyl transferase) α-ketoglutarate dehydrogenase lactate dehydrogenase HMG CoA reductase G-6-P dehydrogenase Answers: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. biotin, biotin pyridoxal phosphate, pyridoxine NAD, nicotinate (or nicotinic acid) FAD, riboflavin TPP, thiamine lipoate (non vitamin) FAD, riboflavin NAD, nicotinic acid NADPH, nicotinic acid biotin, biotin B 12 coenzyme, cobalamin pyridoxal phosphate, pyridoxine coenzyme A, pantothenic acid carnitine (non vitamin), coenzyme A, pantothenic acid same as pyr dehydrogenase NAD, nicotinate NADPH, nicotinate NADP, nicotinate Copyright, L. W. Stillway Revised 2005 Guide to Flying 48 Match the allosteric modifier with the appropriate enzyme: carnitine acyl transferase I phosphofructokinase glucokinase HMGCoA reductase isocitrate dehydrogenase pyruvate dehydrogenase pyruvate carboxylase acetyl CoA carboxylase pyruvate kinase A.. cholesterol B. NADH C. citrate D. glucose 6-phosphate E. malonyl CoA F. acetyl CoA G. palmitoyl CoA H. alanine I. fructose 1,6-bisphosphate J. ATP K. AMP L. none of the above Answers: 1. 2. E. This regulates β -oxidation and ketogenesis by controlling the flux of fatty acids into mitochondria. Sequence: Fatty acyl CoA  Fatty acyl carnitine  fatty acyl CoA C,J, K Citrate produced in the TCA cycle inhibits glycolysis at this point. Citrate is a signal that the TCA cycle has been shut down and not in need of carbons. In addition ATP at high concentrations inhibits by binding to an allosteric site. AMP relieves this inhibition by competing with ATP for the allosteric site. J. Glucokinase is exclusively a liver enzyme and is not regulated allosterically. Since ATP is a substrate, the rate will be concentration-dependent. Hexokinase is found in other tissues, and is regulated by the product G-1-P. A. This is the regulatory enzyme (committed step) for cholesterogenesis and may be inhibited by cholesterol. It may also be regulated genetically, i.e., more or less enzyme protein is synthesized. B,F,J. NADH inhibits as well as ATP. All of this infers a high energy charge for this key step in the TCA cycle. Note that inhibition of this enzyme will result in higher citrate levels that will inhibit PFK-1 and stimulate acetyl CoA carboxylase. AMP stimulates isocitrate dehydrogenase. B,F,J. A high-energy charge decreases the activity of pyruvate dehydrogenase and causes the enzyme to be phosphorylated to an inactive form. F. Acetyl CoA is an obligatory positive modifier of pyruvate carboxylase. This is a signal for more OAA to be produced for: l) stimulation of the TCA cycle, 2) gluconeogenesis because of the utilization of fat and the need for glucose (Fatty acids cannot produce glucose.), 3) The synthesis of fatty acids, ie, citrate must be made to transport acetyl CoA out of mitochondria. C, G. Acetyl CoA carboxylase is the regulatory step in fatty acid synthesis. Citrate stimulates by causing an aggregation of subunits and fatty acyl CoA esters inhibit. H,I,J. High levels of alanine signal the breakdown of extrahepatic proteins (mainly muscle) as part of the glucose-alanine cycle. Since alanine is used for gluconeogenesis, it makes sense to shut off pyruvate kinase so that pyruvate will not be made from PEP. When PFK-1 is stimulated, the production of F-1,6-P stimulates pyruvate kinase to get the last reaction of glycolysis going. ATP inhibits by causing a cAMP-mediated phosphorylation. 3. 4. 5. 6. 7. 8. 9. Copyright, L. W. Stillway Revised 2005 Guide to Flying 49 The red blood cell. 1. 2. 3. 4. 5. 6. 7. What is the only energy-producing pathway in the RBC? Why? This pathway can also produce 2,3-bisphosphoglycerate. Does this compound increase or decrease the affinity of Hb for O2? What would be the effect of a deficiency of pyruvate kinase on BPG and O2 carrying capacity in RBC? What is the effect of decreased pH on O2 binding by Hb? What is this called? What may happen to an individual when glucose 6-P dehydrogenase is deficient and certain drugs such as primaquine, aspirin or sulfa are given? What effect does this have on glutathione and the RBC? Answers: 1. 2. 3. 4. 5. Glycolysis In fact, this is a major purpose for glycolysis in the RBC. BPG (DPG) decreases O2 affinity. More DPG would be produced leading to less O2 carrying capacity. Decreases O2 binding (Bohr effect). NADPH levels are significantly decreased. Glutathione requires NADPH to remain reduced. If NADPH is low, glutathione becomes oxidized and the RBC membrane loses structural integrity and the cells may lyse. Copyright, L. W. Stillway Revised 2005 Guide to Flying 50 Supposing a person is acidotic and large amounts of methylmalonate are found in the urine. 1. 2. 3. 4. 5. 6. 7. What is the cause of the acidosis? What effect does this have on the transport of ? on the ratio of HC03/CO2? What disease is probably the cause? What enzyme defect could also be the cause? The deficiency of which vitamin is usually the cause? How do certain people become deficient in this vitamin? Why does a slight change in pH cause drastic changes in enzyme activities? Answers: 1. 2. 3. 4. 5. 6. 7. Methylmalonic acid is produced because of excess methylmalonyl CoA, which is hydrolyzed to the free acid and coenzyme A. Decreased pH decreases O2 affinity for hemoglobin (Bohr effect). Increased acidity (decreased pH) decreases the HC03/CO2 ratio (metabolic acidosis). pernicious anemia Methylmalonyl CoA mutase converts methylmalonyl CoA to succinyl CoA. B 12 or cobalamin Usually, there is a lack of (or defective) intrinsic factor. This is a glycoprotein secreted in the stomach, and it is required to bind cobalamin. Without intrinsic factor, B12 cannot be absorbed in the intestine under ordinary circumstances. Cobalamin, itself, is poorly absorbed in the intestine, so a person lacking intrinsic factor will become B12 deficient, even though the vitamin is present in the diet in adequate amounts. The extrinsic factor is B12. (Vitamin B12 is stored in the liver, and so little is metabolically required, most of us have a 7-year supply. B12 is not found in plants) Each enzyme has a pH optimum and the curve of pH vs. enzyme activity can be quite steep, so a small change in pH can cause a drastic change in enzyme activity. Copyright, L. W. Stillway Revised 2005 Guide to Flying 51 Conversion of glucose to fat and vice versa. 1. Under what circumstances will the conversion of carbohydrate to fat be stimulated? 2. What intermediate provides the glycerol portion of triacylglycerols (triglycerides)? 3. Is the mitochondrial membrane permeable to acetyl CoA? If not, how is it transported out? 4. May fat be converted to glucose? 5. How might lipolysis stimulate gluconeogenesis? 6. If fatty acids do not yield a net synthesis of carbohydrate, from what compounds is carbohydrate synthesized in the body? 7. The synthesis of fatty acids requires reducing power. What compound provides this reducing capability, and what pathways produce it? Answers: 1. 2. 3. After a meal, resting state Glycerol 3-P from DHAP in glycolysis: The supply of glycerol-3-P controls the rate of synthesis of TG in fat cells, because free glycerol cannot be phosphorylated to glycerol 3-P fat cells. No, coenzyme A derivatives, such as fatty acyl CoA, HMG CoA, etc. cannot cross the inner mitochondrial membrane without some transport mechanism. In the case of acetyl CoA, it is transported out of mitochondria as citrate. Acetyl CoA first condenses with OAA to form citrate, which can traverse the inner mitochondrial membrane, and in the cytosol, citrate lyase cleaves citrate back to acetyl CoA and OAA. Generally, fat (triglycerides) is not a significant source of carbohydrates, because the fatty acids cannot be converted to glucose; however, the glycerol portion of fat can be phosphorylated in the liver to glycerol 3-P, which can be oxidized to DHAP, a gluconeogenic compound. A typical fat molecule contains over 50 carbons, and since glycerol contains only three carbons, it does not contribute significantly to the overall carbon pool. During gluconeogenesis, only about 5% of the glucose is made from glycerol and none from fatty acids. Remember cause and effect? Lipolysis, itself, may not be a major stimulator of gluconeogenesis. However, it is known that high levels of acetyl CoA will allosterically stimulate pyruvate carboxylase. This has the effect of stimulated the conversion of pyruvate to OAA, a key glucogenic intermediate. Recall that the amino groups and carbons of many amino acids released from skeletal muscle end up as pyruvate (glucose-alanine cycle or alanine cycle). (Lipolysis generally occurs at the same time as gluconeogenesis. For example, in starvation, lipolysis and gluconeogenesis are stimulated. The same is true of high-fat and high-protein diets, as well as diabetes and several other diseases, such as von Gierke's disease (glycogen storage disease type I). In all of these, there a combination of hormones, allosteric modifiers and covalent modifications, etc. that simultaneously regulate these pathways. The main thing to remember here is what does not yield a net synthesis of carbohydrate. Acetyl CoA and molecules that yield acetyl CoA do not yield a net synthesis of glucose because of the asymmetric behavior of citrate in the TCA cycle. This would include such compounds as acetoacetate, β-hydroxybutyrate (ketone bodies), leucine and lysine (amino acids). Now to answer the question, most of the glucose is synthesized from amino acids released from skeletal muscle proteins. NADPH is used in reductive biosynthesis, and it is produced by the hexose monophosphate shunt, otherwise known as the phosphogluconate pathway or pentose phosphate pathway. Another source of NADPH is the malic enzyme, which is operating full-tilt during fatty acid biosynthesis. (You may want to check your syllabus or text on how the malic enzyme is related to the transport of acetyl CoA out of mitochondria.) Guide to Flying 4. 5. 6. 7. Copyright, L. W. Stillway Revised 2005 52 Enzymes 1. 2. 3. If an enzyme catalyzes the conversion of A to B, will it increase the concentration of B? What does activation energy have to do with enzyme catalysis? What does it tell you? How does it differ from free energy change? Where does a substrate bind to an enzyme? Is it possible for a substrate to bind to more than one site on an enzyme protein? 4. In the diagram above: a. What part of the curve is said to be linear? b. What is the approximate Km? c. What are the dimensions of Km? d. Where is Vm? e. What accounts for the decreased velocity in the region of C? Would the substrate be a homotropic or heterotropic modifier? Describe induced fit. Differentiate this from allosterism. 5. Answers: 1. No, catalysis does not change the position of equilibrium. It only changes the rate of attainment of equilibrium. 2. An enzyme decreases the activation energy of a reaction, ie; it takes less energy to put the reactants into a transition state. Activation energy tells one something about the rate of a reaction. The free energy change of a reaction gives no information concerning rate; it is only the maximum amount of useful energy that can be obtained from a reaction. 3. Substrates bind to the active site, but they may also bind to an allosteric site (see below). 4. a. The linear portion is part A of the curve. The velocity is very nearly proportional to [S] in this region. b. About one mole/l. Km is the [S] at 1/2 Vm c. It is a concentration term (eg. moles/l). d. At B. e. Substrate inhibition. Substrate is binding to an allosteric site at higher concentrations to inhibit the enzyme activity, ie, as substrate concentration is increased beyond a certain point, it inhibits the enzyme. The allosteric site, in this case, has a lower affinity for substrate than the active site. The substrate would be a homotropic modifier, since the substrate and inhibitor are the same molecule. 5. Induced fit involves a conformational change when substrate binds to an enzyme, making it more active. This usually involves a conformational change in which the protein "wraps around" the substrate, much like a Venus flytrap when it engulfs an insect. Allosterism literally means "other site," so an allosteric modifier binds to a site other than the active site. Copyright, L. W. Stillway Revised 2005 Guide to Flying 53 A B v v [S] In the figures above: 1. Which represents competitive inhibition? Why? 2. What type of inhibition does A represent? [S] Answers: (Note: These are classic Michaelis-Menten curves.) 1. B. In competitive inhibition, the inhibition may be overcome by adding more substrate, which will compete with the inhibitor for the active site. Vm is the same, but Km is increased. 2. A. Non-competitive inhibition. Vm is lower, meaning that inhibition cannot be overcome by more substrate, and Km remains the same. 1. 2. Which of the above Lineweaver-Burke plots represents competitive inhibition? Which represents non-competitive inhibition? Why? Copyright, L. W. Stillway Revised 2005 Guide to Flying 54 Answers: 1. 2. Competitive inhibition - B Non-competitive - A (Use the same logic regarding Vm and Km used in the previous questions.) Glucokinase has a higher Km than hexokinase. 3. What does this mean in terms of the amount of substrate required to achieve the same velocity? Answer: Since Km is the substrate concentration at 1/2 Vm, a higher Km means that a higher concentration of substrate is required by glucokinase to achieve half-maximal velocity. Copyright, L. W. Stillway Revised 2005 Guide to Flying 55 Identify these reactions with a pathway. 1. HOOC 2. O R 3. O HOOC CH CH3 4. O CH3 5. NH2 HOOC 6. O CH (CH2)3 NH CH NH2 HOOC NH2 CH (CH2)3 NH2 C CH2 COOH CH3 OH CH CH2 COOH C SCoA HOOC CH2 CH2 O C SCoA CHOH CH2 C SCoA R O C CH2 O C SCoA CH CH COOH HOOC CHOH CH2 COOH + O H2N C NH2 -O O O CH2 CH OH C H O O O CH2 CH OH C O O P OO O P OO- P O- -O P O- 7. O CH3 8. NH2 HOOC 9. O HOOC C CH2 COOH HOOC O O P OOCH (CH2)3 NH2 O NH2 NH2 HOOC CH (CH2)3 NH O C NH2 C COOH CH3 NH2 CH COOH + 2- O3PO C + GTP CH2 C Copyright, L. W. Stillway Revised 2005 Guide to Flying 56 Answers: 1. 2. 3. 4. 5. 6. 7. 8. 9. TCA cycle, conversion of fumarate to malate β-Oxidation of fatty acids Metabolism of propionate, requires B12 Ketogenesis, acetoacetate, hydroxybutyrate Urea cycle, synthesis of urea from arginine Glycolysis, synthesis of 1,3-bisphosphoglycerate from glyceraldehyde 3-phosphate GPT or alanine transaminase Urea cycle, formation of citrulline from carbamoyl phosphate and ornithine by ornithine transcarbamoylase Gluconeogenesis, regulated step, PEP carboxykinase. Which of the following will result in a net synthesis of glucose? 1. Leucine 2. Palmitic acid 3. Acetoacetic acid 4. Hydroxymethylglutaryl CoA 5. β-Hydroxybutyrate 6. Propionyl CoA 7. Stearoyl CoA 8. Oxaloacete 9. Lactate 10. Glycerol Answers: Only 6, 8, 9 and 10 are potentially gluconeogenic. All the others will yield only acetyl CoA and cannot yield a net synthesis of glucose. Copyright, L. W. Stillway Revised 2005 Guide to Flying 57 1. 2. 3. 4. 5. 6. 7. 8. 9. Answers: 1. 2. 3. 4. 5. In the titration curve above, where is the isoelectric point located? Where is a pK? Is there more than one pK? If so, where is it located? Where does maximum buffering occur? As a rule-of-thumb, what is the pH range where maximum buffering occurs? Where is the fully protonated species located? The zwitterion? The unprotonated species? What type of compound was titrated in the figure? B. The negative and positive charges are equal. A & C. These are points where the least change in pH occurs. At the pK's. One pH unit on either side of a pK or a total of two pH units. D, B, E A monocarboxylic, monoamino acid (glycine) Copyright, L. W. Stillway Revised 2005 Guide to Flying 58