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Gerry Reyna Wiley Plus Assignment #6 The side chain of Glycine is –H. The side chain of Alanine is –CH3 The side chain of Methionine is –CH2-CH2-S-CH3 The side chain of Phenylalanine is –CH2-Phenyl Proline is the only amino acid of the 20 standard amino acids that lacks a primary amine. A peptide bond is a CO-NH linkage that is formed between amino acids. The side chain of Threonine can form a hydrogen bond. The side chain (containing an epsilon-amino group) of Lysine can form a charged ion. The side chain of methionine is NOT aromatic. However, the side chains of Phenylalanine, Tryptophan, and Tyrosine are aromatic. The side chain of Isoleucine is one of the most nonpolar of all the amino acids. Glycine is the only amino acid of the 20 standard amino acids that lacks a chiral center. The image below is a pentapeptide, containing four peptide bonds. Gerry Reyna The peptide Ala-Cys-Gly-Met-Lys has four peptide bonds, two positive charges, and a disulphide bridge. This peptide contains four peptide bonds and two positive charges, one on the Lys side chain and one on the N-terminal amino group. The exclusion of non-polar substances from aqueous solution is the primary driving force in the formation of protein tertiary structure. Irregular secondary structures (loops) are generally found on the surface of globular proteins so that they can interact with the solvent. The peptide groups of the polypeptide backbone in irregular secondary structure do not form hydrogen bonds with one another. Therefore, they are available to form hydrogen bonds with the solvent (water) at the surface of the protein. In regards to the picture below: Protein 2 contains anti-parallel β-strands, and protein 1 contains all three types of secondary structures. Quaternary structure is stabilized by the same types of noncovalent interactions as tertiary structure. Ala, Leu, Phe is the most likely series of amino acids to be buried in the center of a water-soluble globular protein. Ala, Leu and Phe all have nonpolar side chains and so this series is highly likely to be buried in the hydrophobic core of a water-soluble globular protein. Glu-Asp-Lys is the amino acid sequence most likely to be at the surface of a water-soluble globular protein. All three of these amino acids have polar, charged side chains. Structurally myoglobin and hemoglobin are very similar. Their main difference is that myoglobin does not have quaternary structure whereas hemoglobin does. Histidine residues play several critical roles in hemoglobin function. For example, think of the proximal histidine, the distal histidine, and the histidine residues involved in the binding of 2,3- bisphosphoglycerate (BPG). Gerry Reyna The following statements about BPG are true: -BPG requires a binding site containing multiple positively charged groups. -BPG binds to hemoglobin at one site and lowers hemoglobin's affinity for oxygen at another site -BPG binds less tightly to fetal hemoglobin than to adult hemoglobin, thereby aiding oxygen transfer to a fetus -BPG does not affect the affinity of myoglobin for oxygen. In the interaction between an allosteric protein and an allosteric effector, the effector binds reversibly at a specific site on one subunit of the protein, causing a global change in conformation. If a Lys (charged) residue that interacts with 2,3-bisphosphoglycerate (stabilizes the T state) (BPG) in the central cavity of hemoglobin is changed to a Ser residue, the T (tense, deoxygenated) state would be less stable. The replacement of Lys with Ser (not charged) would reduce the affinity of hemoglobin for BPG and the T state would be less stable. Oxygen binding triggers the transition from T state to R state (low to high affinity) in hemoglobin. When oxygen binds to the Fe(II) ion in the heme ring, at its 6th coordination position, the Fe(II) ion is pulled into the plane of the heme ring. It is this binding interaction that initiates the transition from T to R state Hemoglobin's affinity for oxygen is sensitive to small changes in pH (the Bohr effect) because Histidine side chains in hemoglobin become charged at lower pH forming salt bridges that stabilize the T state. Histidine side chains in hemoglobin are more likely to be charged at lower pH and some of these charged side chains can form salt bridges that help to stabilize the T state. (Other histidine side chains, located in the central cavity, increase the binding affinity for BPG at lower pH. Together, these two events confer hemoglobin's sensitivity to changes in pH.) The fact that Myoglobin is a monomeric protein, whereas hemoglobin is a tetrameric protein and that Hemoglobin binds BPG which stabilizes the deoxy (T) state are the most important factors that contribute to their different functions. In the absence of BPG, hemoglobin is unable to adopt its T state. In this situation it binds oxygen with high-affinity only, producing a hyperbolic binding curve. (High pH, no + ions to stabilize it) A newly-identified protein has a sigmoidal curve in a graph of fractional saturation versus ligand concentration. It can be deduced that the protein binds the ligand cooperatively (like hemoglobin). Gerry Reyna The binding of one O2 to a molecule of hemoglobin results in an increased affinity for O2 in the remaining subunits (which have not yet bound O2). In regards to the picture below: Curve 1 is hyperbolic which is typically seen with non-cooperative binding. Curve 3 represents the binding of oxygen to Hb at pH 7.4, thus curve 4 represents the binding of oxygen to Hb at pH 7.2. None of the curves would represent the binding behaviour of a mutant hemoglobin that binds BPG irreversibly Glu Asp would be the most conservative amino acid substitution assuming that these two residues occur at the same position in two homologous proteins. Asp is the most conservative amino acid substitution because, at cellular pH, it involves a change of side chain charge that is opposite (from negative to positive). The following statements about collagen are true: -Prolyl hydroxylase requires ascorbic acid (vitamin C) to maintain its activity. -The modified prolines found in collagen are synthesized from proline post-translationally by an enzyme called prolyl hydroxylase. -Scurvy is a disease caused by vitamin C deficiency that results in weak collagen. -The introduction of vitamin C containing limes to the diet of the British navy alleviated scurvy and led to the nickname “limey” for the British sailor. Gerry Reyna -Collagen includes nonstandard residues like 4-hydroxyproline (Hyp) and 3-hydroxyproline. -Collagen include the nonstandard residue 5-hydroxylysine (Hyl). In the absence of ascorbic acid, prolyl oxidase is unable to oxidize proline residues in collagen to hydroxyproline, resulting in the disease scurvy. Collagen is a triple-helical fibrous protein. Wiley Plus Assignment #7 In order to catalyze reactions, enzymes frequently require additional substances in their active site known as cofactors. Cofactors include substances classified as coenzymes, prosthetic groups, and essential ions. The difference in free energy between the substrate and product of a reaction catalyzed by Enzyme A is negative and small. It can be concluded that the reaction is spontaneous and its speed is unknown from this data. In the picture below ΔG represents the overall free energy change of the catalyzed reaction. Enzymes decrease the activation energy of the reaction they catalyze which is why they can increase the rate of a reaction. Gerry Reyna Enzymes can be regulated because they are proteins. The catalytic ability of enzymes depends on their precise 3-D conformation, and proteins are able to change their conformation. Given figure 1 and 2 below, it can be concluded that figure 1 presents a spontaneous reaction while figure 2 does not. Many enzymes change shape upon substrate binding. Enzymes catalyze reactions at their active site. Enzymes form complexes with their substrates. Enzymes do not alter the free energy of the reaction they catalyze. The His side chain is able to act as both an acid and a base under physiological conditions which is the best explanation for the frequent appearance of His side chains in enzyme active sites. The pKa of the His side chain enables it to act reversibly as an acid and a base under physiological conditions. This unique property of His side chains is frequently used by enzymes in acid-base catalytic mechanisms. The role of an enzyme in a biological reaction is to increase the rate at which substrate is converted to product. Enzymes that perform oxidation-reduction reactions are called oxidoreductases. Enzymes that perform group elimination reactions to form double bonds are called lyases. Gerry Reyna The line labeled X in the graph below is representative of the free energy of the transition state. Enzymes work by reducing the activation energy barrier of a reaction. The following processes contribute to this. 1) The enzyme ensures appropriate proximity and orientation for substrates at its active site. 2) The enzyme binds the substrate specifically and with high affinity 3) Specific amino acid side chains in the active site play a part in the catalytic mechanism. The enzyme does NOT bind the co-substrate covalently. A well-designed enzyme active site provides all of the following features: A. Complementarity of shape and chemical nature to the substrate(s). B. Proximity and correct orientation of substrates and catalytic groups. C. Complementarity of shape and chemical nature to the transition state The region of an enzyme where catalysis occurs is the active site. The point of highest free energy on the reaction coordinate is called the transition state. Gerry Reyna Capacity for regulation and a high degree of substrate specificity are two characteristics of enzyme- catalyzed reactions. Molecules that differ from the substrate in shape or in distribution of functional groups cannot bind productively to the enzyme. This best explains the concept of geometric and electronic complementarity between enzyme and substrate. An ionized sulfhydryl group could act as a nucleophile in an enzyme. Metal ions participate in the catalytic process by binding to substrates to orient them properly for reaction. Lysine is the amino acid in an enzyme that could be responsible for an observed enzyme catalyzed reaction at pH 9. Some serine proteases are believed to have developed by convergent evolution, because the amino acid sequences of some serine proteases show no resemblance to those of others. Clustering several amino acid residues with favorable pK values at an active site can promote a concerted acid-base catalytic mechanism. Methyl is an amino acid group that would not make a good nucleophilic catalyst. The imidazole side chain of histidine can function as either a general acid catalyst or a general base catalyst because in the physiological pH range, the nitrogen in the ring can be easily protonated/deprotonated. Leucine residues would not provide a side chain for acid-base catalysis. Isoleucine residues would provide a side chain capable of increasing the hydrophobicity of a binding site. Elastase is a serine protease that has a specificity pocket that binds small hydrophobic side chains. Serine proteases use covalent catalysis, proximity and orientation catalysis, general base catalysis, and electrostatic catalysis to catalyze the cleavage of a peptide bond. Chymotrypsin catalyzes the hydrolysis of peptide bonds adjacent to large nonpolar residues, amide and ester bonds, and reagents such as p-Nitrophenolate p-Nnitrophenylacetate. Gerry Reyna Some functions of enzymes include the addition or removal of a substance to a substrate and the rearrangement of a substrate the alteration of the reaction temperature. Wiley Plus Assignment #8 The graph illustrates the activity of various enzymes, where the rate of the reaction (v) is plotted as a function of substrate concentration. In regards to the graph: Curve 1 is hyperbolic which is typical of a non-allosteric enzyme. The Absence of activator is indicated by curve 3; the presence of an activator is indicated by curve 2. The following mechanisms are known to play a role in the reversible alteration of enzyme activity. 1. Allosteric response to a regulatory molecule. 2. Covalent modification of the enzyme. 3. Interactions between catalytic and regulatory subunits. 4. Alteration of the synthesis or degradation rate of an enzyme. Gerry Reyna The plateau in the graph below indicates that the enzyme is working close to its maximal activity under the experimental conditions. That is, the active site is saturated. The following statements about allosteric enzymes are true: 1. The substrate of an allosteric enzyme may also be an allosteric effector for that enzyme. 2. Allosteric enzymes have tertiary structure. 3. Allosteric effectors elicit a change in shape of the enzyme via a process termed cooperativity. The following statements about enzymes are true: 1. Enzymes are often very specific for their substrates. 2. Enzymes catalyze reactions in both directions. 3. Enzyme activities can often be regulated. 4. Enzymes typically catalyze reactions at much higher rates than chemical catalysts. Gerry Reyna 5. Enzymes typically act under milder conditions of temperature and pH than chemical catalysts. Given this reaction: We know that [ES] is constant. The Michaelis constant KM is defined as (k–1 + k2)/k1 Transition state analogs often make better competitive inhibitors than do substrate analogs. Activation by cleavage of an inactive zymogen by proteolytic cleavage is irreversible. In competitive inhibition the inhibitor binds reversibly at the active site. An allosteric inhibitor decreases the activity of an enzyme by binding to a site other than the active site. The most efficient enzymes have k_cat/K_M values near the diffusion -controlled limit. Enzymes that bind reaction transition states with greater affinity than substrates or products are inhibited by transition-state analogs. A lead compound would be most promising if it had a KI = 1.5 x 10-8 M. KM is the [S] that half-saturates the enzyme. An enzyme is considered to have evolved to its most efficient form if kcat/KM is near the diffusion- controlled limit. Parallel lines on a Lineweaver-Burk plot are diagnostic of non-competitive inhibition. A compound that reduces the concentration of enzyme available for substrate binding is called a competitive inhibitor. Allosteric activators bind to enzymes and stabilize the 'R-state', which has an enhanced substrate affinity.
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