Enzymes Regulation Aspartate Transcarbamoylase

ENZYMES: A. Enzyme Mechanisms 1. Proteases 2. Serine Proteases a. Reaction & specificity b. Activation (zymogens); Ile a-amino pKa somehow important c. Active Site Determination d. Proposed mechanism e. Specificity 3. Other protease mechanisms B. Enzyme Regulation 1. Introduction 2. Strategies a. Gene Regulation b. Covalent Modification c. Allosteric Control 3. Covalent Modification a. Proteolysis b. Protein modification i. Phosphorylation a) Kinases b) Phosphatase c) Example-PKA 4. Allosteric Control a. Regulation nomenclature i. On/off switch ii. Example: ATCase b. Review; binding curves; why sigmoidal c. Hill Equation d. Physical models; 3° and 4° conf. changes i. To vs. Ro ii. Sequential; KNF iii. Concerted (symmetry); MWC iv. Examples of measuring these parameters Control by Allostery: Enzyme Regulation Allosteric Effectors can change binding/kinetic behavior EXAMPLE: Aspartate Transcarbamoylase (ATCase) 1 Control by Allostery: Enzyme Regulation Pyrimidine Biosynthesis: ATCase Controlled by Classic Feedback Inhibition Control by Allostery: Enzyme Regulation Allosteric Effectors: ATCase Reaction ON OFF CTP is a negative heterotropic allosteric effector molecule ATP is a positive heterotropic allosteric effector molecule 2 Control by Allostery: Enzyme Regulation Allosteric Effectors can change binding/kinetic behavior Other examples: Physiological concentration Physiological concentration Physiological concentration Control by Allostery: Enzyme Regulation Why has sigmoidal kinetics evolved? • Not just to create ON/OFF switches • Maintain homeostasis If [S] decreases, the rate of its use goes down. Eventually, the [S] is replenished. This acts to “buffer” the activity at around ½Vmax, often right at the homeostatic [S]. AàSàPàQ If [S] increases, the rate of enzyme goes up, and S gets used up. Eventually, the [S] is lowered. Physiological concentration of substrate. 3 Control by Allostery: Enzyme Regulation What is the degree of cooperativity? Michaelis-Menton Equation Like binding, we can also express as fraction of maximum rate Recall for cooperative binding, there is an exponent term on the [S]. What is K’m term? n0 = Vmax[S] Km +[S] n0 = [S] Vmax Km +[S] n0 = Vmax The K’m term is a mixture of values for both poor(at low [S]) and high-affinity (at higher [S]) binding sites. What is the value of this n’ term? Control by Allostery: [S]n’ K’m +[S]n’ The n’ term is related to the degree of cooperativity. Enzyme Regulation In 1913, Archibald Hill derived an equation to measure both the K’m term, and the n’ term: The Hill Equation Fraction of [E]T as [ES] (Bound) Fraction of [E]T as [E] (Free) Related to = Y 1–Y = n0 Vmax = [S]n’ K’m +[S]n’ n’ 1 – [S] n’ K’m +[S] [S]n’ K’m m +[S]n’) m +[S]n’) x (K’ (K’ Take log of both sides Sometimes you will see: n’ = h, the Hill coefficient log ( 1 –Y Y ) = n’ log [S] – log K’m (Eqn for line à y = ax + b) This has the form of a line with n’ as the slope and –logK’m as the xintercept in a plot of log Y/(1-Y) versus log [S]. This is a “Hill Plot.” 4 Control by Allostery: Enzyme Regulation The Hill Plot log Y (1–Y ) = n’ log [S] – log K’m Y Theoretical maximum cooperativity = # of binding sites 0 Positive cooperativity: n > 1 Control by Allostery: Non-cooperative: n = 1 at low and high [S] Negative cooperativity: n < 1 Enzyme Regulation Physically, how does this cooperativity work? In other words, how is this cooperativity accomplished at the molecular level? Recall that sigmoidal behavior requires more than one subunit. Therefore, subunits must “communicate,” or binding of one subunit changes the subunit-subunit interface, and this changes how the non-bound subunits will bind. EXAMPLE: • Dimer • Two conformations Ø unbound enzyme = T (tense) state Ø bound enzyme = R (relaxed) state • Conformation at interface differs • Binding site differs with binding easier (tighter) in the R-state 5 Control by Allostery: Enzyme Regulation Two ways have been proposed to explain the transition for Tstate to R-state binding. This type of cooperativity would be homotropic (caused by the substrate itself) All at once = CONCERTED [Proposed by Monod, Wyman, & Changeux (MWC)] Control by Allostery: One at a time = SEQUENTIAL [Proposed by Koshland, Nemethy, & Filmore (KNF)] Enzyme Regulation If it binds to the T-state, it would be an inhibitor. (negative heterotropic allosteric effector) Heterotropic cooperativity can be achieved by binding at a different allosteric site. If it binds to the R-state, it would be an activator. (positive heterotropic allosteric effector) 6 Control by Allostery: Enzyme Regulation How about for a tetramer? Sequential Model of cooperative regulation • S does not bind to the T-state. • Binding of ligand to one subunit changes its conformation. • That conformational change influences neighboring subunits through changes at the subunitsubunit interface. • The binding to the neighboring subunits is easier (Kd decreases). • Model has many different binding and dissociation constants (very complicated rate equations). Control by Allostery: Enzyme Regulation Concerted Model of cooperative regulation e • Looks more complicated, but its simpler in concept and mechanism • An equilibrium exists between the T-state and the R-state • Binding of substrate is easier to the R-state • Binding of substrate to one subunit shifts the equilibrium. • That shift brings neighboring subunits, which are also high affinity; more ”good” sites. 7 Control by Allostery: Enzyme Regulation Two Models of Cooperativity: Concerted vs. Sequential All at once = CONCERTED [MWC] Control by Allostery: One at a time = SEQUENTIAL [KNF] Enzyme Regulation We can write the rate equation for the Concerted Model of cooperative regulation: The equilibrium between the T-state and the R-state = [T0]/[R0] = L Binding of substrate is easier to the R-state, which is reflected in a ratio of dissociation constants = c = KR / KT (less than 1.0). A rate equation can be derived using these values: For a dimer….. [S] (n-1) [S] (1 + KR ) KR n0 = kcat [ES] = [ES] = Bound S = Y = [S] (n) Vmax = kcat [E]T Total sites [E]T L + (1 + KR ) For dimer, exponent in denominator If L=0 (no T-state) ….... [S] [S] KR Recall eqn for coop: = Y = n0 = [S]n’ [S] KR + [S] (1 + KR ) Vmax K’m +[S]n’ 8 Control by Allostery: Enzyme Regulation si 9 Is there structural evidence for these T- and R-states? Enzyme Regulation Control by Allostery: R-state Catalytic subunits (a) Regulatory subunits (b) Aspartate Transcarboxylase (ATCase) PALA (a6b6) T-state (a3)2(b2)3 CTP 9 Control by Allostery: Enzyme Regulation ATCase: Conformational Changes: T-State vs. R-State Enzyme Regulation Control by Allostery: Aspartate Transcarboxylase (ATCase) 10 10/25/23 Enzyme Regulation Key Concepts for Control of Enzyme Activity •Allosteric effectors bind to multisubunit enzymes, such as aspartate transcarbamoylase, thereby inducing cooperative conformational changes that alter the enzyme’s catalytic activity (T and R states). •Phosphorylation and dephosphorylation of an enzyme such as glycogen phosphorylase by protein kinases can control its activity by shifting the equilibrium between more active and less active conformations (T and R states). You should be able to: • Compare and contrast the actions of an allosteric effector, a competitive enzyme inhibitor, and a noncompetitive inhibitor. • Explain the structural basis for cooperative substrate binding and allosteric control in ATCase. • Why are such allosteric enzymes composed of more than one catalytic subunit? • Distinguish between KNF and MWC models for cooperativity and how homotropic and heterotropic effectors act • Understand what can be derived from a Hill Plot 11