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5,7,8,10,13,15,17,20,21 Recognition of Substrates by Enzymes

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5,7,8,10,13,15,17,20,21 Recognition of Substrates by Enzymes Recommended problems from chapter 15: 5,7,8,10,13,15,17,20,21 Recognition of substrates by enzymes See chemical mechanisms notes on web page, covered in chapter 14 in your textbook. Also see chapter 13 for discussion of the “lock and key” vs. the “induced fit” substrate binding modes. 2 hypotheses on how substrates fit within the active site of enzymes: 1. “Lock and key” •Exact fit between substrate and enzyme 2. “Induced fit” •Enzyme active site is “flexible” or “dynamic” and can assume distinct (but probably related) conformations. A corollary is that it can probably accommodate distinct (but probably somewhat similar) substrates. • “Good” substrates can fit into the active site in such a way that the transition state is approximated. 1 General considerations in the regulation of enzymes 1. Genetic control. At this level of regulation the amount of the enzyme is changed in response to environmental conditions. For example, in response to an abundance of glucose, the production of metabolic enzymes that cleave large polysaccharides stops. In the case of lac repressor, this regulation is on the transcription level. 2. Control of enzymatic activity. At this level the amount of an enzyme is not varied. Rather, the activity of the enzyme is regulated (up or down). A. For S ↔ P, as the amount of product builds up the reverse reaction becomes more pronounced, until equilibrium is reached, where there is no net change in the amounts of product and substrate. B. Enzymatic rates can be modulated by the amount of available substrates and their Km values. C. Covalent modification can alter the affinity of enzyme to substrate (alter the Km). Phosphorylation and de-phosphorylation are such control systems. Nobel prize in 1992 was awarded to Edmond Fischer and Edwin Krebs for this discovery. D. Allosteric effectors are molecules that alter the affinity of an enzyme to a substrate. These are non-covalent modifications. Regulation of enzyme activity by phosphorylation Phosphorylation can occur on serine, threonine, or tyrosine residues. What are the predicted physical consequences of this modification on an enzyme active site? 2 Aspartate transcarbamoylase as a model system of allosteric control The reaction catalyzed by ATCase is diagrammed below. This is one of the initial reactions in pyrimidine synthesis. O O- C NH2 CH2 + C C 2- O OPO3 + - H3N COO H Carbamoyl phosphate Aspartic acid aspartate transcarbamoylase O O- C NH CH - 2 2 + H2PO4 C C O N COO- H H N-Carbamoyl aspartate Feedback inhibition of ATCase by CTP 3 Allosteric models •Allosteric interactions affect the activity of an enzyme by binding at a site distinct from the active site (allos = different; stereo = place or solid). •Allosteric effectors can be the substrate(s); this is a homotropic effect. •The effector(s) can be distinct from the substrate(s) molecules; this is a heterotropic effect. •An allosteric effector can be positive (increases enzymatic activity) or negative (decreases enzymatic activity). Often both effects are found for a given enzyme. For example, in the hemoglobin system: O2 is a homotropic positive effector (increases affinity for oxygen at the oxygen binding site). Bisphosphoglycerate (BPG) and CO2 are negative heterotropic effectors (decrease affinity for oxygen). In general, allosteric effects result from interactions among subunits of oligomeric proteins. It is possible, however, that single-subunit proteins can exhibit allosteric effects if there are multiple binding sites for substrates within the single polypeptide. The Monod, Wyman, and Changeux allosteric model: cooperative binding Consider a protein that may assume two states. The “taut” state binds substrate very poorly, whereas the “relaxed” state has a high affinity for substrate. The subscript 0 denotes an unbound protein, and these two states exist in equilibrium, such that: R0 ↔ T0 Further assume that the equilibrium of R0 and T0 lies substantially to the right, such that in the absence of substrate there is a lot more taut form than relaxed. If L is the association equilibrium constant, L = [T0] / [R0] and L is large. If KR is the equilibrium dissociation constant of substrate from the relaxed form, and KT is the equilibrium dissociation constant of substrate from the taut form, then by the conditions above KR << KT. The extreme case is KR / KT = 0, and in this case there is no affinity of T0 to substrate. 4 The Monod, Wyman, and Changeux allosteric model: cooperative binding R0 ↔ T0 If substrate is added to this system, the equilibrium is perturbed. R0 binds the substrate and no longer exchanged with T0. Now the reaction above goes to the left, more R0 is made, and, in turn, more substrate is bound by R0, and so on. This is a positive homotropic effect. The Monod, Wyman, and Changeux allosteric model: cooperative binding c = KR / KT ; L = [T0] / [R0]; n = number of substrate binding sites; Y = [filled substrate binding sites] / [total number of substrate binding sites]. This substrate binding behavior is cooperative. Filling of a binding site alters the apparent overall affinity for substrate. All binding sites, however, are equivalent. 5 The Monod, Wyman, and Changeux allosteric model; positive heterotropic effects Each subunit has an allostery site and a substrate binding site. If an allosteric activator (A) is present, then the R0 ↔ T0 reaction goes to the left, and there are more substrate binding sites available. This equilibrium shift results in an increase of the apparent (measured) affinity for substrate at a fixed activator concentration. Overall, the cooperativity of substrate binding decreases, because there are more substrate binding sites available. The allosteric activator has the same effect as a lower equilibrium constant L. The Monod, Wyman, and Changeux allosteric model; negative heterotropic effects In the presence of inhibitor, the equilibrium R0 ↔ T0 goes to the right. This reduces the number of substrate binding sites available, and lowers the apparent affinity for substrate. In the presence of a fixed concentration of inhibitor, a titration of substrate would show an increase in the cooperativity of binding, but a decrease in the apparent affinity. 6 The Monod, Wyman, and Changeux allosteric model; heterotropic effects Plot of v0 versus [Aspartate] for the ATCase reaction Is ATP an inhibitor or activator? How does it affect cooperativity of aspartate binding? Is UTP an inhibitor or activator? How does it affect cooperativity of aspartate binding? Does this make sense if you consider the role of ATCase in pyrimidine synthesis? 7 Structure of E. coli ATCase •A view along the 3-fold axis of the 300 kDa protein complex. Composition is c6r6. •The 6 catalytic subunits are in red and blue, and consist of 2 trimers stacked atop each other. •The 6 regulatory subunits are in yellow, there are 3 dimers that bind the two trimers. •Each trimer has catalytic activity that is higher than the catalytic activity of the complex. The trimer is neither affected by ATP nor CTP. What does this argue about the role of the regulatory subunits? Where do you expect the regulatory allostery effectors to bind? Structure of E. coli ATCase The T-state conformation The R-state conformation A view perpendicular to the previous slide, along the molecular two-fold symmetry axis. Given the information about ATCase in the previous slides, what reagent would you add to the protein in order to obtain these two distinct structure? 8 Structure of E. coli ATCase The T-state conformation The R-state conformation O O PALA is a compound that C C PO 2- Carbamoyl binds the enzyme but 3 H N C O PO 2- H2 2 3 phosphate does not react. NH + NH3 Predict to which -OOC C C COO- - - H2 H OOC C C COO Asp artate conformation PALA H2 H would bind. N-(Phosphonaetyl)-L-aspartate (PAL A) The Koshland, Nemethy, and Filmer model, AKA the sequential model •The symmetry model assumes a constant, fixed substrate binding site (Emil Fischer’s “lock and key” model). •In contrast, the sequential model allows for changed in the conformation of the other binding sites as a result of substrate binding at a particular site on the oligomeric protein. The binding sites are NOT always equivalent. •This means that substrate binding sites are “coupled,” such that filling one binding site changes the other binding sites on the oligomeric protein. •The degree of “coupling” among the subunits can vary. At the extreme, when the subunits are completely coupled, the sequential model actually resembles the symmetry model. 9 The symmetry model of allostery •A tetramer protein is represented. •All the subunits of a given protein can be either in the T or the R form. •No combinations of R and T subunits are allowed within the protein. •The T and R states are at equilibrium regardless of the number of substrates bound. The sequential model of allostery •Binding of S to one subunit of the enzyme can induce a conformational change in the other subunits of the enzyme. •The greatest change occurs at the subunits that have bound ligand. •The symmetry of the protein subunits is not preserved as was the case with the symmetry model. •At the heart of this model are the distinct conformations that the subunits assume as a consequence of substrate binding. 10 Hemoglobin as a model system for allosteric regulation Hemoglobin and myoglobin serve similar functions. They bind O2 and deliver it as needed. Hemoglobin is found in blood, whereas myoglobin is found in muscle. Each polypeptide binds a single heme group, a porphyrin ring structure that binds Fe2+. The porphyrin ring system also binds O2. Oxygen binding by myoglobin If the heme group contains Fe2+, oxygen binding is efficient. In contrast, if the heme group contains Fe3+, oxygen binding is poor.
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