Enzyme Regulation Aspartate Transcarbamoylase

Enzyme Regulation “Enzyme” Regulation: Hemoglobin 1. Introduction 2. Strategies 1. Roles of Hb a. Gene Regulation b. Covalent Modification Xx c. Allosteric Control 1. 2. Xx 3. Covalent Modification 1.Proteolysis 2.Protein modification i. Phosphorylation a) Kinases b) Phosphatase 1. 2. 3. X X X c) Example: PKA 4.Allosteric Control a.Regulation nomenclature a. Oxygen transport b. CO2 binding c. Blood buffer: Bohr effect 2. Oxygen Binding/role of protein 3. Binding curves a. b. c. d. oxygen Allosteric effectors (BPG) Bohr effect; (protons) Carbon dioxide 4. Structure-Function; Structural basis for physiology (T & R states) 5. Mechanism of Cooperativity i. 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 Feedback Inhibition Control by Allostery: Enzyme Regulation Allosteric Effectors: ATCase Reaction 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? 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 it 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 The Hill Plot log Y (1–Y ) = n’ log [S] – log K’m Y Theoretical maximum 0 Positive cooperativity: n > 1 Non-cooperative: n = 1 at low and high [S] cooperativity = # of binding sites Negative cooperativity: n < 1 5 Control by Allostery: 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 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)] One at a time = SEQUENTIAL [Proposed by Koshland, Nemethy, & Filmore (KNF)] 6 Control by Allostery: 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. Control by Allostery: If it binds to the R-state, it would be an activator. (positive heterotropic allosteric effector) 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). 7 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. Control by Allostery: Enzyme Regulation Two Models of Cooperativity: Concerted vs. Sequential All at once = CONCERTED [MWC] One at a time = SEQUENTIAL [KNF] 8 Control by Allostery: 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: = K + [S] Y= n0 = [S]n’ [S] R (1 + KR ) Vmax K’m +[S]n’ Control by Allostery: Enzyme Regulation si 9 Is there structural evidence for these T- and R-states? 9 Enzyme Regulation Aspartate Transcarboxylase (ATCase) Control by Allostery: R-state PALA Catalytic subunits (a) Regulatory subunits (b) (a6b6) T-state (a3)2(b2)3 CTP Control by Allostery: Enzyme Regulation ATCase: Conformational Changes: T-State vs. R-State 10 Enzyme Regulation Control by Allostery: Aspartate Transcarboxylase (ATCase) 11