Lecture 19 (10/30/17) Enzyme Regulation

Transcription

1 Reading: Ch5; 164, Problems: none Remember Today at 6:30 in PHO-206 is the first MB lecture & quiz NEXT Reading: Ch5; , , Lecture 19 (10/30/17) Problems: Ch5 (text); 3,7,8,10 Ch5 (study guide-facts); 1,2,3,4,5,8 Ch5 (study guide-apply); 2,3 Remember Wednesday at 5:30 in CAS-522 is the second chance for the MB lecture & quiz ENZYMES: A. 1. Covalent Modification 2. Allosteric Control a. Regulation nomenclature 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 B. Enzyme Regulation: Hemoglobin 1. Roles of Hb a. Oxygen transport b. CO 2 binding c. Blood buffer: Bohr effect 2. Oxygen Binding/role of protein 3. Binding curves a. oxygen b. Allosteric effectors (BPG) c. Bohr effect; (protons) d. Carbon dioxide 4. Structure-Function; Structural basis for physiology (T & R states) 5. Mechanism of Cooperativity Control by Covalent Modification: EXAMPLE: Protein Kinase A a 2 b 2 (90 kda & 40 kda) a is Regulatory (R) b is Catalytic (C) and has the kinase activity The active site recognizes a particular site on substrate proteins Target sites have the consensus sequence: R R XSB Inactive Active Why is PKA inactivate when bound to R small 2? R has a sequence: RRGAI that fits into the C active site! How is this equilibrium shifted? Cyclic-AMP (camp) K Large hydrophobic 1

2 What is camp? H H2O phosphodiesterase For PKA, camp is known as a positive heterotropic allosteric effector What is Allostery or Allosteric Control? (meaning: allo = another, steric = place, site, space) Control by binding to another site; NOT the active site Nomenclature for allosteric effector molecules: molecules that are the substrate = homotropic molecules that are the not the substrate = heterotropic molecules that activate the enzyme = positive molecules that inhibit the enzyme = negative Hence, camp is a positive heterotropic allosteric effector for PKA 2

3 Recall binding curves: R + L k 1 k -1 R L K D = [R][L] [R L] Fraction bound (B) (Y) = [R L] [R] T [ L] Y = K [ L] D + Y Y 1.0 on d 0.5 Single kind of binding site site Multiple kinds of binding sites D Ligand concentration [L] WHY IS THIS SIGMOIDAL BEHAVIOR IMPORTANT FOR REGULATION? IF AFFORDS A WAY TO MAKE AN ON/OFF SWITCH Example of famous binding proteins: 3

4 Allosteric Effectors can change binding/kinetic behavior +positive allosteric effector Non- allosteric enzyme (M-M enzyme) +negative allosteric effector [S] If this is physiological concentration of substrate, we have an ON/OFF switch! Allosteric Effectors can change binding/kinetic behavior Non- allosteric enzyme (M-M enzyme) Rate with M-M kinetics (+positive effector) +positive allosteric effector Rate with positive heterotropic allosteric effector (or sometimes these positive effectors convert the enzyme to a non-cooperative kinetics (M-M)). Rate with Sigmoidal kinetics +negative allosteric effector Rate with negative heterotropic allosteric effector [S] If this is physiological concentration of substrate, we have an ON/OFF switch! From book: At increasing conc., positive effectors (blue) or negative effectors (red) can affect the Km or the Vmax to achieve the same switch. 4

5 Allosteric Effectors can change binding/kinetic behavior EXAMPLE: Aspartate Transcarbamoylase (ATCase) Pyrimidine Biosynthesis: ATCase Feedback Inhibition 5

6 Allosteric Effectors: ATCase Reaction Aspartate Transcarboxylase (ATCase) Catalytic subunits (a) Regulatory subunits (b) (a 3 ) 2 (b 2 ) 3 CTP ATCase from E. coli PDBids 5AT1 and 8ATC 6

7 Allosteric Effectors can change binding/kinetic behavior Other examples: Physiological concentration Physiological concentration Physiological concentration Why has sigmoidal kinetics evolved? If [S] decreases, the rate of its use goes down. Eventually, the [S] is replenished. If [S] increases, the rate of its use goes up. Eventually, the [S] is lowered. This acts to buffer the activity at around ½V max, often right at the homeostatic [S]. Physiological concentration of substrate. 7

8 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]. n 0 = V max [S] K m +[S] n 0 = [S] V max K m +[S] n 0 = [S] n V max K m +[S] n What is K m term? 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? The n term is related to the degree of cooperativity. Fraction of [E] T as [ES] (Bound) Fraction of [E] T as [E] (Free) Related to n 0 V max In 1913, Archibald Hill derived an equation to measure both the K m term, and the n term: The Hill Equation [S] n K m +[S] n Sometimes you will see: n = h, the Hill coefficient Y = 1 Y ( ) = = 1 [S] n K m [S] n K m +[S] n x Y log = n log [S] log K m 1 Y (Eqn for line à y = ax + b) This has the form of a line with n as the slope and logk m as the x- intercept in a plot of log Y/(1-Y) versus log [S]. This is a Hill Plot. (K m +[S] n ) (K m +[S] n ) Take log of both sides 8

9 The Hill Plot Y log( 1 Y ) = n log [S] log K m Y 0 Theoretical maximum cooperativity = # of binding sites Positive cooperativity: n > 1 Non-cooperative: n = 1 Negative cooperativity: n < 1 at low and high [S] The Hill Plot Y log( 1 Y ) = n log [S] log K m Y 0 Theoretical maximum cooperativity = # of binding sites Positive cooperativity: n > 1 Non-cooperative: n = 1 Negative cooperativity: n < 1 at low and high [S] 9

10 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 Two ways have been proposed to explain the transition for T- state 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)] 10

11 If it binds to the T-state, it would be an inhibitor. (negative heterotropic allosteric effector) Heterotropic cooperativity can be achieved by binding at an allosteric site. If it binds to the R-state, it would be an activator. (positive heterotropic allosteric effector) 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 (K d decreases). Model has many different binding and dissociation constants (very complicated rate equations). 11

12 Concerted Model of e cooperative regulation 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, but empty. Two Models of Cooperativity: Concerted vs. Sequential 12

13 We can write the rate equation for the Concerted Model of cooperative regulation: The equilibrium between the T-state and the R-state = [T 0 ]/[R 0 ] = L Binding of substrate is easier to the R-state, which is reflected in a ratio of dissociation constants = c = K R / K T (less than 1.0). A rate equation can be derived using these values: For a dimer.. [S] n 0 = k cat [ES] [ES] Bound S K R = = V max = k cat [E] = Y = T [E] T Total sites L + For dimer, exponent in denominator If L=0 (no T-state)... [S] Recall eqn for coop: n 0 = [S] n V max K m +[S] n Y = K R = [S] K R (1 + ) [S] K R + [S] [S] (1 + ) K R [S] (1 + ) K R (n-1) (n) si 9 13

14 ATCase: Conformational Changes: T-State vs. R-State ATCase: Conformational Changes: T-State vs. R-State 14

15 Glycogen Phosphorylase: Control by Allostery & Phosphorylation (more active) Glycogen (n) + P i + Glycogen (n-1) Glycogen Phosphorylase: Control by Allostery & Phosphorylation (more active) Glycogen (n) + P i + Glycogen (n-1) 15

16 Rabbit Muscle Glycogen Phosphorylase (dimer) Covalent phosphoryl group (activating) Allosteric positive effector Notice that these are both near the subunit-subunit interface and both add a phosphoryl group 45 This is the R-state. Is there are T-state? Active site Rabbit Muscle Glycogen Phosphorylase AMP Phospho-Ser Glycogen phosphorylase PDBids 8GPB and 7GPB Active site 16

17 Rabbit Muscle Glycogen Phosphorylase AMP Phospho-Ser Glycogen phosphorylase PDBids 8GPB and 7GPB Active site 17