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Enzyme chemistry of nucleophilic oxy anion and enzyme-coenzyme chemistry of electron and group transfer I. Purpose and principle of enzyme catalysis: 1. Why biocatalysis is needed and how it is done. 2. What enzymes can and cannot do. 3. Shape fitting a prerequisite to enzyme specificity and catalysis. 4. A practical and revealing description of enzyme kinetics. 5. Kinetics of an allosteric enzyme that shows cooperativity. 6. Chemical background of catalysis by general-acid/base and metal ion. II. Enzyme chemistry of nucleophilic oxy anion: 1. Serine protease oxy anion as nucleophile. Triad catalytic strategy is an especially effective approach to peptide hydrolysis, Structure of chymotrypsin, Selective peptide cleavage, Chymotrypsin and related hydrolytic enzymes (trypsin, elastase) and their substrate specificities, Papain S¯ in lieu of O¯ as nucleophile. 2. Aspartyl protease of HIV and protease inhibitor as AIDS drug. Mechanism of specific inhibition of HIV protease. 3. Carboxypeptidase A cleaves peptide bond next to C-terminal-COO¯ using Zn++. 4. More Zn++ ion catalysis and synthetic model of carbonic anhydrase. 5. Lysozyme hydrolyzes bacterial cell wall composed of β-glycosidic linkages. 6. Isomerization of dihydroxyacetone phosphate to glyceraldehyde 3-phosphate in glucose catabolism. 7. Bacterial transpeptidase. Penicillin inactivates an enzyme-serine in bacterial cell-wall synthesis. III. Enzyme-coenzyme chemistry of electron transfer (NAD+ and FAD): 1. Coenzyme and vitamins for electron transfer and structural link. 2. Structures of NAD+/NADH & niacin vitamin for redox reactions. 3. Glyceraldehyde 3-phosphate dehydrogenase and NAD+. NAD+-binding region in dehydrogenases, Catalytic mechanism, Free energy profiles for glyceraldehyde oxidation coupled/uncoupled with acyl- phosphate formation. 4. Flavin redox coenzymes FAD/FADH2 are covalently bound to enzyme and complimentary to NAD/NADH. Mechanism of dehydrogenation, Why FAD as another electron carrier and why not use NAD+ throughout, FAD precedes NAD+ in major metabolic pathways. IV. Enzyme-coenzyme chemistry of group transfer (Imine chemistry of pyridoxal and cryptic carbanion chemistry of thiamin): 1. Coenzyme and vitamins for group transfer. 2. Pyridoxal in group transfer reactions. Structures of pyridoxal (free and enzyme bound) and reactivity, Pyridoxal mediate group transfers, Mechanism via imine-enamine intermediates (transamination, decarboxylation, transfer of RCHO, transfer of -H makes L-amino acids), Seleative bond cleavage by stereoelectronic control of PLP Enzymes. 3. Cryptic carbanion chemistry of thiamin. Structure of thiamin and unique ylidene as carbanion, Significant role of thiamin in mediating between glycolysis and the citric acid cycle, pyruvate dehydrogenase complex joins with thiamin for crucial link, Mechanism via thiamin ylidene intermediate in decarboxylation and transacetylation for the dehydrogenase system, Schematic diagram of the dimer interface environment around the catalytic center, Illustration of complete reaction schemes of the dehydrogenase complex, Detailed picture of the transketolase-thiamin-substrate, Fundamental structural questions resolved. I. Purpose and principle of biocatalysis 1. Why biocatalysis is needed. Fast reactions are required to sustain life. For example, the half-life for chemical hydrolysis of a peptide bond at neutral pH is 10 - 1000 years. Yet, peptide bonds must be hydrolyzed within milliseconds in some biochemical processes. See the table of rate enhancement achieved as enzyme catalysis has evolved. Rate enhancement by selected enzymes Enzyme Nonenzymatic Rate enhancement half-life (kcat/kun) OMP decarboxylase 78,000,000 years 1.4 × 1017 Staphylococcal 130,000 years 5.6 × 1014 nuclease Carboxypeptidase A 7.3 years 1.9 × 1011 Triose phosphate 1.9 days 1.0 × 109 isomerase Carbonic anhydrase 5 seconds 7.7 × 106 (OMP, orotidine monophosphate) A. Radzicka and R. Wofenden. Science 267 (1995):90 93. How it is done. The enzymes (or with coenzymes) help to form and stabilize the transition state. The basic strategies are: (1) Bring reacting groups together in the active site, properly oriented, to achieve specificity. (2) Exert binding energy to stabilize the TS. (3) Form more active intermediate, e.g., amide to acid product via ester. (4) Use side chains of amino acids for general acid-base catalysis. (5) Metal ion, when bound to enzyme, participates in coordination and as a Lewis acid. (6) Make use of coenzymes for H¯ and C¯ involvement. Six major classes of enzymes Class Type of reaction Example 1. Oxidoreductases Oxidation-reduction Lactate dehydrogenase 2. Transferases Group transfer Nucleoside monophosphate kinase 3. Hydrolases Hydrolysis reactions (transfer of functional groups Chymotrypsin to water) 4. Lyases Addition or removal of groups to form double bonds Fumarase 5. Isomerases Isomerization (intramolecular group transfer) Triose phosphate isomerase 6. Ligases Ligation of two substrates at the expense of ATP Aminoacyl-tRNA synthetase hydrolysis 2. What enzymes can and cannot do Enzymes alter only the reaction rate and not the equilibrium. Consider The equilibrium concentration of B is 100 times that of A, whether or not enzyme is present. Enzyme accelerates both forward and reverse rates, hence attaining equilibrium faster but do not shift its K. K = function of ∆G between reactants and products. Catalysis What enzymes can do: facilitates TS formation by lowering its activation energy TS energy lowered by enzyme cat. Catalyst destabilizes the transition state entice Hall 2001 Chapter 22 4 3. Shape fitting a prerequisite to enzyme specificity and catalysis Much of the catalytic power of enzymes comes from bringing substrates together in optimal orientations at the active site. Formation of the enzyme-substrate (ES) complexes is a prelude to making and breaking bonds at TS. Enzymes contain >100 amino acids, MW >10 kd and a diameter of >25 Å. The vast majority of amino acids serve as a scaffold to create the 3-D active site, a pocket, from a few amino acids that may be far apart in the primary structure. Enzymes are flexible and adopt conformations that are structurally and chemically complementary to the transition states of the reactions that they catalyze. Thus, other than lock and key fit, shape fitting of ES complex may be induced. Substrates are bound in the active site by multiple weak attractions: electrostatic, H-bonds, van der Waals, and hydrophobic forces to give ES complexes with K that range from 10 -2 to 10-8 M. This binding energy of the ES complex helps to stabilize the TS. 4. A practical and revealing description of enzyme kinetics A plot of the reaction velocity (V0) as a function of the substrate concentration [S] for an enzyme shows the maximal velocity (Vmax) is approached asymptotically. Note KM, a constant, is the [S] yielding a velocity of Vmax/2. KM lies between 10-1 and 10-7 M, pointing to [S] required for significant catalysis to occur. KM is also a measure of the strength of the ES complex: a high KM indicates weak binding; a low KM indicates strong binding. This behavior is summarized in the Michaelis-Menten equation: Substrate concentration when low, [S] << KM, V0 = (Vmax/KM)[S]; rate is directly proportional to [S]. When high, [S] >> KM, V0 = Vmax; rate is maximal, independent of [S]. Physiological consequence of KM: Consider the sensitivity of some individuals to ethanol. Such persons exhibit facial flushing and rapid heart rate after ingesting even small amounts of alcohol. In the liver, CH3CHO is the cause of intoxication, then [O] to CH3COO¯ by acetaldehyde dehydrogenase. Most people have 2 forms of the acetaldehyde dehydrogenase, a low KM mitochondrial form and a high KM cytosolic form. In susceptible persons, the mitochondrial enzyme is less active due to a single amino acid mutation, leaving CH3CHO to the cytosolic enzyme. Because this enzyme has a high KM, less CH3CHO is converted into CH3COO¯; excess CH3CHO escapes into the blood and accounts for the physiological effects. Meaning of Vmax: The maximal rate reveals the turnover number, which is the number of substrate molecules converted into product by an enzyme molecule in a unit time. e.g., A 10-6 M solution of carbonic anhydrase catalyzes the formation of 0.6 M H2CO3 s-1 when fully saturated with CO2. Hence, turnover is 6 × 105 s-1, one of the largest known. The opposite is 0.5 s-1 for lysozyme, but most enzymes fall in the range from 1 - 104 s-1. 5. Kinetics of an allosteric enzyme that shows cooperativity Allosteric enzymes display a sigmoidal dependence of reaction velocity on [S]. Here the binding of substrate is cooperative (binding of substrate to one active site facilitates substrate binding to the other active sites). Allosteric effect of enzyme Consider hexokinase which binds glucose in the first step of glycolysis. The result of this step is to “prime” glucose for subsequent breakdown by attaching a phosphate to it. This phosphate comes from ATP, which binds to hexokinase after the binding of glucose causes a change in the enzyme’s shape. Thus, hexokinase can exist in two forms, depending on what is bound. This is allosterism or “other forms”. In this case, the allosteric change is one that activates the enzyme. Another allosteric scheme is (1) the change from binding ATP, and (2) the next change when the ATP is hydrolyzed. This scheme is the motor driving the muscle protein, myosin, and ion pumps. The allosteric enzyme activity may be altered by regulatory molecules bound to sites other than the catalytic sites, e.g., hemoglobin binding O2 is cooperative and is regulated by H+, CO2 etc. 6. Chemical background of catalysis Electron push and pull most effective cid Catalysis If the proton transfer occurs during the is known slow step, it catalyzed hydrolysis of ester as general-acid Chemical examples of catalysis and involves a weak acid, HA Intramolecular Catalysis General-acid catalysis Putting the reacting group and the catalyst on the same molecule has an effect similar General-base catalysis to placing both reacting groups on the same molecule Intramolecular Cata Ortho-carboxyl as intramolecular general-base catalyst: aspirin susceptible to moisture ice Hall 2001 Chapter 22 12 The or substit intramo Acid Catalysis genera that inc nucleo © Prentice Hall 2001 Chapter 22 25 water This is called intramolecu catalysis or anchimeric a © Prentice Hall 2001 Chapter 22 Specific cat. (activated species preformed before TS) is more for chemical than bio reactions. © Prentice Hall 2001 Chapter 22 13 Metal-Ion Catalysis A metal ion can Metal ion catalysis stabilize a transition state byA metal ion can receiving make a leaving electrons from a group a weaker center where a base negative charge is developing Metal-Ion Catalysis TS stabilized Leaving tendency of OCH3 enhanced © Prentice Hall 2001 Chapter 22 18 © Prentice Hall 2001 Chapter 22 19 Better nucleophile A metal ion can complex with water Water becomes more acidic when metal-bound, yielding an effective HO¯ and increase its acidity © Prentice Hall 2001 Chapter 22 20 II. Enzyme chemistry of nucleophilic oxy anion 1. Serine protease oxy anion nucleophile: Triad catalytic strategy is an especially effective approach to peptide hydrolysis. Catalytic triad found in chymotrypsin: Asp 102 side chain induces His 57 to remove H+ from Ser 195, thus activating it to Ser–CH2O¯ to initiate peptide hydrolysis. Structure of chymotrypsin (endopeptidase). 3 chains are shown in ribbon form in orange, blue, and green. The side chains of the catalytic triad residues, including serine 195, unusually reactive, are shown. Chymotrypsin is inactivated by treatment with diisopropylphosphofluoridate (DIPF), which reacts only with serine 195 among 28 possible serine residues. Selective peptide cleavage Chymo cleaves peptide bonds selectively on the carboxyl terminal side of the large hydrophobic amino acids such as try, tyr, phe, and met. Hydrolysis takes place in two stages: (A) acylation to form the acyl-enzyme intermediate followed by (B) deacylation to regenerate the free enzyme. Chymotrypsin and related hydrolytic enzymes (trypsin, elastase) and their substrate specificities The sequences of trypsin and elastase are approximately 40% identical with that of chymotrypsin, and their overall structures are nearly the same. An overlay of the structure of chymotrypsin (red) on that of trypsin (blue) shows the high degree of similarity. Only a-carbon atom positions are shown. The mean deviation in position between corresponding a-carbon atoms is 1.7 Å. Different substrate specificities: Trypsin cleaves peptide bond after residues with long, positively charged side chains (Arg, Lys). Elastase cleaves peptide bond after amino acids with small side chains (Ala, Ser). Papain S¯ in lieu of O¯ as nucleophile Papain, an enzyme from the papaya fruit. 2. Aspartyl protease of HIV and protease inhibitor as AIDS drug Human immunodeficiency virus (HIV) and other retroviruses contain an unfused dimeric aspartyl protease. The central feature of the active sites is a pair of aspartic acid residues that act together to allow a water molecule to attack the peptide bond. Electron push and pull model: Crixivan, an HIV protease inhibitor, is used in the treatment of AIDS. This protease cleaves multidomain viral proteins into their active forms; blocking this process prevents the virus from being infectious. HIV Protease-Crixivan Complex The flaps close down on the binding pocket after substrate has been bound. Mechanism of specific inhibition of HIV protease Crixivan is constructed around an alcohol that mimics the tetrahedral intermediate. The OH group of the central alcohol interacts with two aspartate residues of the active site, one in each subunit. In addition, two carbonyl groups of the inhibitor are H-bonded to H2O, which, in turn, is H-bonded to a peptide NH group in each of the flaps. This interaction of the inhibitor with water and the enzyme is not possible with cellular aspartyl proteases such as renin and thus may contribute to the specificity of Crixivan. chanism for 3. Carboxypeptidase A cleaves peptide bond next to C-terminal-COO¯ using Zn++ rboxypeptidase A (an exopeptidase) sm for all 2001 st Chapter 22 30 1 Step: molecular recognition by Tyr 248 and Arg 145, 127; hydrophobic pocket H O Nu A peptidaseenhanced by Glu 270 general-base cat. & Zn cat. (3 aa coord.) 2 2+ ng ged t on ine Mechanism for Carboxypeptidase A second step, In the 22 Chapter 31 Glu 270 functions as a general-acid catalyst increasing the leaving tendency of the amino group Note only few of the proteinases work as amino- or carboxypeptidases, while most are endopeptidases © Prentice Hall 2001 Chapter 22 35 preferably cleaving peptide bonds within a polypeptide chain rather than at its ends. 4. More Zn++ ion catalysis and synthetic model of carbonic anhydrase CO2, a major end product of aerobic metabolism, is released into blood and transported to the lungs for exhalation. It is hydrated in the blood to carbonic acid, and dissociates H+ (pKa = 3.5) to bicarbonate. This hydration proceeds at 0.15 s-1; the reverse 50 s-1, hence K = 5.4 × 10-5, giving a ratio of [CO2] to [H2CO3] of 340:1. Carbonic anhydrases accelerate CO2 hydration dramatically. The human carbonic anhydrase II can hydrate CO2 at ~106 s-1. This is required because CO2 hydration and HCO3¯ dehydration are often coupled to rapid processes like transport processes or eye secretions. Human carbonic anhydrase II and Its Zinc Site. (Left) The zinc is bound to the imidazole rings of three histidine residues as well as to a water molecule. (Right) The location of the zinc site in the enzyme. Mechanism of action Histidine Proton Shuttle. (1) His 64 abstracts H+ from Zn++-HOH, generating a nucleophilic ¯OH and His-H+. (2) The buffer (B) removes a proton from His-H+, regenerating the unprotonated form. Synthetic model accelerates hydration of carbon dioxide more than 100-fold Step 1, pKa 7 Mechanism for Lysozyme 5. Lysozyme hydrolyzes bacterial cell wall composed of -glycosidic linkages In lysozyme, a -glycosidase that degrades the cell walls of some bacteria, the active site is made up of residues 35, 52, 62, 63, 101, and 108 in the sequence of the 129 amino acids As Lysozyme binds the substrate, it change shape slightly, distorting the sugar residue site D, into a half-chair Mechanism for Lysozyme destabilizes the reactant, relative to th This transition state © Prentice Hall 2001 Chapter 22 54 Lysozyme hydrolyzes bacterial cell wall © Prentice Hall 2001 Chapter 22 Mechanism: Asp 52 electrostatic cat. (salt bridge), Glu 35 general-acid cat. Then general-base cat. on HOH D-ring distorted into half-chair towards TS conformation, favoring glycosidic cleavage. max. activity at pH 5.3 (Asp as anion, Glu as acid, surrounded by nonpolar groups) 6. Isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde 3-phosphate (GAP) This reaction in glycolysis is rapid and reversible. At equilibrium, 96% of the triose phosphate is DHAP, but reaction proceeds readily from DHAP to GAP because the subsequent reactions of glycolysis remove this product. Triose Phosphate Isomerase (TIM) catalyzes this reaction. Two notable points about TIM (1) TIM, a kinetically perfect enzyme, accelerates isomerization by a factor of 1010 compared with that by acetate. The k of rate-limiting step is 2 × 108 M-1 s-1, indicating almost diffusion-controlled encounter of substrate and enzyme. (2) TIM suppresses an undesired side reaction, from enediol to methyl glyoxal. In solution, this elimination is 100 times as fast as isomerization. Hence, TIM must prevent the enediol from leaving the enzyme. This intermediate is trapped in the active site by the movement of a loop of 10 residues. This loop serves as a lid on the active site, shutting it when the enediol is present and reopening it when isomerization is completed. The undesirable alternative reaction is suppressed by having the phosphate group anchored. Structure of Triose Phosphate Isomerase. This enzyme consists of a central core of eight parallel strands (orange) surrounded by eight helices (blue). This structural motif, called an barrel, is also found in the glycolytic enzymes aldolase, enolase, and pyruvate kinase. His 95 and glu 165, essential components of the active site of triose phosphate isomerase, are located in the barrel. A loop (red) closes off the active site on substrate binding. 7. Bacterial transpeptidase. Penicillin inactivates an enzyme-serine in bacterial cell-wall synthesis Bacterial cell walls are unique in containing D amino acids, which form cross-links of different peptidoglycan strands catalyzed by glycopeptide transpeptidase.. The amino group at one end of a pentaglycine chain attacks the peptide bond between two D-alanine residues in another peptide unit. Sugars are shown in yellow, tetrapeptides in red, and pentaglycine bridges in blue. The cell wall is a single, enormous, bag-shaped macromolecule because of extensive cross-linking. Penicillin inhibits the cross-linking transpeptidase disguised as a Trojan horse. The transpeptidase normally forms an acyl intermediate leading to cross-link formation. Penicillin gets into the active site of the transpeptidase because it mimics D-Ala-D-Ala: Penicillin, with a highly strained and reactive 4-mem -lactam, reacts with the transpeptidase to form an inactive complex, which is indefinitely stable. III. Enzyme-coenzyme chemistry of electron transfer: NAD+ and FAD 1. Coenzyme and vitamins for electron transfer and structural link The enzyme function for H¯ transfer requires an electron carrier as coenzyme. The 20 amino acids of protein are ill-equipped to handle H¯ which is too basic and unstable. Coenzymes are small organic molecules derived from vitamins. Since H¯ denotes an electron lone pair and H+, H¯ transfer is also lone pair transfer and hence a redox reaction. Coenzymes required in enzyme functions and the corresponding vitamins Vitamin Coenzyme Typical enzyme Consequences of deficiency function Nicotinic acid Nicotinamide adenine Oxidation- Pellagra (dermatitis, depression, (niacin) dinucleotide (NAD+) reduction diarrhea) Riboflavin (B2) Flavin adenine dinucleotide Oxidation- Cheliosis and angular stomatitus (FAD) reduction (lesions of the mouth), dermatitis These are water-soluble vitamins. Vitamins are needed in small amounts in the diets of higher animals that lost the capacity to synthesize them in the course of evolution. The biosynthetic pathways for vitamins can be complex; thus, it is biologically more efficient to ingest vitamins than to synthesize the enzymes required to construct them. Note Vitamin C, a well-known antioxidant of its own right, is not a coenzyme. In the synthesis of 4- hydroxyproline, an amino acid of the connective tissue collagen, Vitamin C is required for the continuing activity of the reaction enzyme prolyl hydroxylase. This is a dioxygenase with a tightly bound Fe2+ to activate O2. The result is Fe3+ in the inactivated enzyme which is reduced by ascorbate to Fe2+. Structural link: Adenosine Diphosphate (ADP) is a fundamental building block in NADH, FAD, coenzyme A, and ATP. The adenine is shown in blue, ribose in red, and diphosphate in yellow. Adenine C5H5N5, a pentamer of HCN, and ribose have prebiotic origin. These ancient structures are conserved for molecular recognition in various metabolic functions. oxidations, the NAD+/NADH ratio is high ~1000 Anabolic reactions are predominantly reductions - NADP+/NADPH ratio is low ~ 0.01 Niacin: The Vitamin Needed fo 2. Structures of NAD/NADH & niacin vitamin for redox reactions Redox © Prentice Hall 2001 Chapter 23 5 Niacin: The Vitamin Needed for Redox Niacin: The Vitamin Needed for Redox Much of the structural complexity of these tice Hall 2001 Chapter 23 Much of the structural complexity of these recognition coenzymes is for molecular coenzymes is for molecular recognition The business end of NAD+/NADP+ is the The business end of NAD+/NADP+ is the pyridine ring pyridine ring © Prentice Hall 2001 Chapter 23 6 Niacin: The Vitamin Needed for Redox © Prentice Hall 2001 Chapter 23 6 NADPH is used almost exclusively for reductive biosyntheses (anabolism), whereas The NAD +/NADH couple is (catabolism). The phosphoryl group on NADH is used primarily for the generation of ATP used almost exclusively in catabolic reaction whereas the NADP tag that enables couple is used in anabolic NADPH is a+/NADPHenzymes to distinguish between when to use what. reactions Since catabolic reactions are predominantly oxidations, the NAD+/NADH ratio is high ~1000 Anabolic reactions are predominantly reductions - NADP+/NADPH ratio is low ~ 0.01 © Prentice Hall 2001 Chapter 23 5 3. Glyceraldehyde 3-Phosphate dehydrogenase and NAD+ NAD+-binding region in dehydrogenases. In glycolysis and the subsequent conversion of pyruvate, the three dehydrogenases (glyceraldehyde 3-phosphate dehydrogenase, alcohol dehydrogenase, and lactate dehydrogenase) have in common a domain for NAD+ binding. This nucleotide-binding region is made up of 4 helices and a sheet of 6 parallel strands, often called a Rossmann fold. This fold is likely a primordial dinucleotide-binding domain that recurs in the dehydrogenases. The nicotinamide-binding half (yellow) is structurally similar to the adenine-binding half (red). The NAD+ molecule binds in an extended conformation. Structure of Glyceraldehyde 3-Phosphate Dehydrogenase. The active site includes a cysteine residue and a histidine residue adjacent to a bound NAD+. Catalytic mechanism of glyceraldehyde 3-phosphate dehydrogenase. The reaction proceeds through a thioester intermediate, which allows the oxidation of glyceraldehyde to be coupled to the phosphorylation of 3-phosphoglycerate. Why are the two reactions coupled? Advantages are seen by comparing with the activation energies when oxidation and acyl phosphate formation are uncoupled. Free-Energy Profiles for Glyceraldehyde Oxidation Followed by Acyl-Phosphate Formation. (A) A hypothetical case with no coupling between the two processes. The second step must have a large activation barrier, making the reaction very slow. (B) The actual case with the two reactions coupled through a thioester intermediate. Flavin Adenine Dinucleotide and Flavin Mononucleotide 4. Flavin redox coenzymes FAD/FADH2 are covalently bound to enzyme and complimentary to NAD/NADH Mechanism of dehydrogenation Flavin Adenine Dinucleotide and Flavin Mononucleotide There are multi e-addition sites in FAD: S attacks C4a, C at N5 as shown for oxidation ¯ ¯ of amino acid to an imine (net removal of 2 H•) Enzymes that use oxidizing coenzymes other than NAD+ may still require NAD+ to reoxidize the reduced coenzyme © Prentice Hall 2001 Chapter 23 12 © Prentice Hall 2001 Chapter 23 11 Why FAD as another electron carrier and why not use NAD+ throughout Along this line, would it not be even simpler to use molecular oxygen. In the citric acid cycle, O2 does not participate directly. True, the cycle operates only under aerobic conditions because NAD+ and FAD can be regenerated only by electron transfer to O2. Look at succinate oxidized to fumarate by succinate dehydrogenase. The hydrogen acceptor is FAD rather than NAD+, which is used in the other three oxidation reactions in the cycle. In succinate dehydrogenase, a histidine side chain is covalently attached to C-8 methyl of the tricyclic ring of FAD (denoted E-FAD). FAD is the hydrogen acceptor because the free-energy change is insufficient to reduce NAD+. FAD is nearly always the electron acceptor in oxidations that remove two hydrogen atoms from a substrate. Such difference is also seen in the generation of ATP (e-transport chain in the cycle yields a proton-motive force): ~ 2.5 ATP per NADH, and 1.5 ATP per FADH2. Succinate dehydrogenase contains iron-sulfur clusters, 2Fe-2S, 3Fe-4S, and 4Fe-4S. FADH2 produced by the oxidation of succinate does not dissociate from the enzyme, in contrast with NADH produced in other redox reactions. Rather, 2 electrons are transferred from FADH2 directly to iron-sulfur clusters of the enzyme and ultimately to O2. FAD precedes NAD+ in major metabolic pathways Below shows major metabolic pathways with similar sequences of reactions in common: oxidation, hydration, and another oxidation, resulting in methylene group (CH2) converted into a carbonyl group (C=O). Oxaloacetate is regenerated for another round of the cycle, and more energy is extracted in the form of FADH2 and NADH. IV. Enzyme-coenzyme chemistry of group transfer: Imine chemistry of pyridoxal and cryptic carbanion chemistry of thiamin 1. Coenzyme and vitamins for group transfer Vitamin Coenzyme Typical enzyme function Consequences of deficiency Pyridoxine Pyridoxal Group transfer to or from Depression, confusion, convulsions (B6) phosphate amino acids Thiamine Thiamin Aldehyde transfer Beriberi (weight loss, heart problems, (B1) pyrophosphate neurological dysfunction) Other examples of group transfer not discussed here Biotin Biotin-lysine complexes ATP-dependent carboxylation and Rash about the eyebrows, muscle (biocytin) carboxyl-group transfer pain, fatigue (rare) Folic Tetrahydrofolate Transfer of one-carbon components; Anemia, neural-tube defects in acid thymine synthesis development B12 5 -Deoxyadenosyl Transfer of methyl groups; Anemia, pernicious anemia, cobalamin intramolecular rearrangements methylmalonic acidosis These are water-soluble vitamins. Pyridoxal Phosphate In each of these reactions, one of the bonds to the -carbon of the amino acid 2. Pyridoxal in group transfer reactions is broken in the first step Pyridoxal Phosphate © Prentice Hall 2001 Chapter 23 Structures of pyridoxal (free and enzyme bound) and reactivity 28 The most important functional group on PLP is the aldehyde. It allows PLP to form covalent Schiff-base with a specific lysine -amino group of the enzyme or with the amino acid substrates. These Schiff-base linkages are often protonated, with the positive charge stabilized by interaction with the negatively charged phenolate group of PLP. © Prentice Hall 2001 Chapter 23 26 Pyridoxal mediate group transfer in Pyridoxal Phosphate © Prentice 2001 © Prentice HallHall 2001 Chapter 2323 Chapter 26 26 Pyridoxal Phosphate H transfer makes racemization possible © Prentice Hall 2001 Chapter 23 27 rentice Hall 2001 Chapter 23 27 Mechanism via imine-enamine intermediates: transamination, decarboxylation, transfer of RCHO Pyridoxal Phosphate Glutamate (repeat steps above) Pyridoxal Phosphate Transfer of RCHOH as RCHO TransferofH makes L-amino acids © Prentice Hall 2001 Chapter 23 31 In a transaminase active site, addition of H+ from the lysine residue to the bottom face of the quinonoid intermediate determines the L configuration of the amino acid product. The conserved arginine residue interacts with the -carboxylate group and helps establish the appropriate geometry. Seleative bond cleavage by stereoelectronic control of PLP Enzymes PLP enzymes labilize one of three bonds at the -carbon of an amino acid substrate. For example, bond a is labilized by aminotransferases, bond b by decarboxylases, and bond c by aldolases (such as threonine aldolases). How done? An important principle is that the bond being broken must be perpendicular to the orbitals of the electron sink. An aminotransferase achieves this goal by binding the amino acid substrate so that the C- H bond is perpendicular to the PLP ring. e.g., Aspartate Aminotransferase. The active site has a PLP Schiff-base linkage with lysine 268. An arginine helps orient substrates by binding to their -carboxylate groups. In aspartate aminotransferase, the C-H bond is most nearly perpendicular to the -orbital system and is cleaved. In serine hydroxymethyltransferase, the N-C bond is rotated so that the C-C bond is most nearly perpendicular to the plane of the PLP ring, favoring its cleavage. Thiamine Pyrophosphate 3. Cryptic carbanion chemistry of thiamin Structure of thiamin and unique ylidene as carbanion Thiamine Pyrophosphate hiamine Pyrophosphate The hydrogen bonded to imine The hydrogen bonded to thethe imine carbon is relatively acidic, pK = 12.7 carbon is relatively acidic, pKa = a12.7 pKa ~13 © Prentice Hall 2001 Chapter 23 13 Significant role of thiamin in mediating between glycolysis and the citric acid cycle Pyruvate produced by glycolysis is converted into acetyl CoA, the fuel of the citric acid cycle. e Hall 2001 Prentice Hall 2001 Chapter 23 Chapter 23 14 14 Pyruvate dehydrogenase complex of E. coli joins with thiamin for crucial link Enzyme Abbrevn # chains Prosthetic group Reaction catalyzed Pyruvate dehydrogenase E1 24 TPP Oxidative decarboxylation of pyruvate component Dihydrolipoyl transacetylase E2 24 Lipoamide Transfer of the acetyl group to CoA Dihydrolipoyl dehydrogenase E3 12 FAD Regeneration of the oxidized form of lipoamide Schematic Representation of the Pyruvate Dehydrogenase Complex The transacetylase core (E2) is shown in red, the pyruvate dehydrogenase component (E1) in yellow, and the dihydrolipoyl dehydrogenase (E3) in green. The conversion of pyruvate into acetyl CoA consists of three steps: decarboxylation, oxidation, and transfer of the resultant acetyl group to CoA. Pyruvate is decarboxylated at the active site of E1, forming the substituted TPP intermediate, and CO2 leaves as the first product. This active site lies within the E1 complex, connected to the enzyme surface by a 20-Å-long hydrophobic channel. Note these steps are coupled to preserve the free energy derived from the decarboxylation step to drive the formation of NADH and acetyl CoA. Thiamine Pyrophosphate Mechanism via thiamine ylidene intermediate in decarboxylation & transacetylation Thiamine Pyrophosphate The resulting intermediate easily can Thiamine Pyrophosphate decarboxylation as electrons undergo The ylide carbanion left behind can be delocalized onto the attacks the ketone group of an -keto acid (pyruvate) positively charged nitrogen Thiamine Pyrophosphate © Prentice Hall 2001 Chapter 23 15 © Prentice Hall 2001 Chapter 23 16 © Prentice Hall 2001 Chapter 23 17 © Prentice Hall 2001 Chapter 23 20 Schematic diagram of the dimer interface environment around the catalytic center. Bonding interactions between cofactors and protein are indicated by dashed lines. Residues numbered >360 are in the domain of one monomer, while those <187 are in the domain of the 'other" monomer. Illustration of complete reaction schemes of the Pyruvate Dehydrogenase Complex At the top (center), the enzyme (represented by a yellow, a blue, and two red spheres) is unmodified and ready for a catalytic cycle. (1) Pyruvate is decarboxylated to form the hydroxyethyl TPP. (2) The dihydrolipoyl arm of E2 moves into the active site of E1. (3) E1 catalyzes the transfer of the two-carbon group to the dihydrolipoyl group to form the acetyl-lipoyl complex. (4) E2 catalyzes the transfer of the acetyl moiety to CoA to form the product acetyl CoA. The disulfhydryl lipoyl arm then swings to the active site of E3. E3 catalyzes (5) the reduction of the lipoic acid and (6) the transfer of the protons and electrons to NAD+ to complete the reaction cycle. Detailed picture of transketolase-thiamine-substrate Cleavage of the donor substrate D-xylulose 5-phosphate by yeast transketolase Fundamental structural questions resolved J. Phys. Chem. A 2005, 109, 7606-7612 Topological Analysis of the Electron Density in Model Azolium Systems for Thiamin Structure-Function: Sulfur Is the Electron Sink and Positively Polarized Carbanions Act as Nucleophiles Donald B. DuPre´*,† and John L. Wong*,‡ Department of Chemistry, University of Louisville, Louisville, Kentucky 40292 Receivaed: March 22, 2005; In Final Form: June 20, 2005 The mechanism of thiamin diphosphate-dependent enzyme reactions requires two carbanion intermediates 1a and 1b. Neither has been isolated nor detected, but resonance stabilization is assumed to occur with the thiazolium quaternary nitrogen being the electron sink. We have questioned the electronic nature of these carbanion intermediates and, in a broader sense, the role of sulfur in the thiazolium moiety. To address these issues the theory of atoms in molecules (AIM) was used to acquire quantitative electron distributions in thiazolium 2, oxazolium 3, and imidazolium 4 as cations and zwitterions. Among the heteroatoms, only sulfur acts as an electron sink. This is corroborated by a similar behavior in phosphorothioates. Further, the formal carbanion at C2 and Ca of the intermediates are positively charged and their nucleophilic character is explained with AIM theory by comparison with the C- of model 5a and C- of model 6a. C2 of 2a excels in lone pair coverage in the -plane, surpassing the C- in acyclic 5a and other cyclic ylidenes, and hence, is a more effective nucleophile. The C- of 6a reveals a depletion area centered in the -plane but shows lone-pair concentration above and below the plane. Unlike 6a, the AIM properties, bond length, and bond order of 2b indicate no lone-pair on C but essentially a double bond across C2-C. Thus, the nucleophilic behavior at C of 1b is based on the enamine chemistry induced by an electrophile. SCHEME 1: Thiamin Diphosphate Intermediates Contour plot of the Laplacian of the electron density showing regions of charge concentration in a plane perpendicular to the 5- membered ring and approximately bisecting the N3-C2-S1 bond angle of the thiazolium ylidene 2a.
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