BMB Lecture 7. Allostery and Cooperativity

Transcription

1 BMB Lecture 7 October 18, 2017 Allostery and Cooperativity A means for exquisite control

2 Allostery: the basis of enzymatic control From the Greek: allos = other stereos = solid or space Action at a distance Examples of allostery: enzyme cooperativity induced fit covalent regulation feedback regulation oligomerization gating

3 Biology is replete with positive and negative cooperativity Phosphorylation PKA GTPases ATPase motors Channels Receptors Translation bacterial chemotaxis ATP synthase Calcium signaling

4 Simple Ligand Binding [E] total = [E] + [ES] Fraction of ligand bound

5 Hyperbolas are sigmoidal on a semi-log scale

6 Highly cooperative ligand binding Hill coefficient

7 Requiring more than one thing to happen tends to sharpen the dose-response Examples: counting quanta of something arrival times for kinetic processes enzyme activation (e.g., substrate cooperativity)

8 Hemoglobin >> Myoglobin 4

9 Cooperative Oxygen Binding in Hemoglobin O 2 binding curve much steeper than myoglobin (cooperativity) shifts with ph (the Bohr effect) myoglobin Hemoglobin at ph 7.6 (lung) Hemoglobin at ph 7.2 (tissue)

10 Adair constants for O 2 binding to hemoglobin etc. Why are all the K s different if this is a symmetric enzyme?

11 Coupled Equilibria Equilibrium is pathwayindependent Therefore 1 fi 2 fi 3 equals 1 fi 4 fi 3 Reciprocity: binding of substrate promotes conformational change conformational change promotes substrate binding

12 Monod-Wyman-Chamgeux (MWC) concerted mechanism Protein is an oligomer Two reversible conformational states -R state has a higher affinity / activity -T state has a lower affinity / activity Conformation in each protomer is constrained by quaternary interactions => concerted conf. change associated with changes in quaternary structure All binding sites in each conf. are equivalent (symmetry assumption)

13 MWC Binding Curve produces a lovely, sigmoidal binding curve requires only three free parameters symmetry assumption cannot account for negative cooperativity

14 Beautifully Reproduces O 2 binding curve in hemoglobin Monod et al, J. Mol. Biol. 12: 88 (1965)

15 Theoretical Curves from MWC model Cooperativity occurs when conformational equilibrium needs to shift T fi R Get higher cooperativity when T and R states differ significantly in activity Monod et al, J. Mol. Biol. 12: 88 (1965)

16 Prediction 1. Allosteric activator reduces cooperativity, allosteric inhibitor does the opposite g = activator shifts equilibrium T fi R b = inhibitor shifts equilibrium R fi T Monod et al, J. Mol. Biol. 12: 88 (1965)

17 Effect of allosteric activators on substrate binding Isocitrate dehydrogenase AMP- activator Deoxythymidine kinase Monod et al, J. Mol. Biol. 12: 88 (1965)

18 Prediction 2a: substrate makes binding of allosteric inhibitors more cooperative Threonine deaminase [Isoleucine] Allosteric inhibitor Monod et al, J. Mol. Biol. 12: 88 (1965)

19 Prediction 2b: Allosteric activator makes inhibitor binding more cooperative [isoleucine] Monod et al, J. Mol. Biol. 12: 88 (1965)

20 Prediction 3: Competitive substrate inhibitors can stimulate reaction Aspartate Transcarbamylase maleate - competitve inhibitor Monod et al, J. Mol. Biol. 12: 88 (1965)

21 Koshland-Nemethy-Filmer (KNF) Model asymmetic model conformation of each subunit is altered in turn conformational change is mainly local (3 not 4 ) conformational change is transmitted to neighboring subunits If substrate affinity decreases, can account for negative cooperativity # free parameters = # binding sites

22 Multiple Binding Sites (Adair Equation)

23 MWC vs. KNF

24 MWC vs. KNF Predict different intermediate states. Both work exceedingly well for positive cooperativity. MWC has fewer parameters, and is more elegant. MWC cannot account for negative cooperativity, but KNF can. Both are serious simplifications.

25 Eigen s Model (n+1) 2 states, 2n(n+1) K s, and O(n 2 ) constraint equations

26 Structural basis for cooperative oxygen binding to Hemaglobin oxy deoxy

27 O 2 binding induces changes in Heme binding site

28 Phosphofructokinase: key allosteric regulation during glycolysis Binding of F6P is sigmoidal Allosteric activation by ADP Allosteric inhibition by PEP

29 Crystal structure of PFK R-state (ADP bound) A effector site B Active site Schirakihara & Evans, JMB 204, 973 (1988)

30 Crystal structure of PFK T-state (PGC bound) A effector site C Active site B D Schirmer & Evans, Nature 343: 140 (1990)

31 Structural basis for allosteric behavior of PFK Schirmer & Evans, Nature 343: 140 (1990)

32 Interactions across subunit interface (r) T-state R-state Schirmer & Evans, Nature 343: 140 (1990)

33 Movement of Active Site T-state R-state Schirmer & Evans, Nature 343: 140 (1990)

34 Negative Cooperativity glyceraldehyde-3-p dehydrogenase Binding of each DPN weakens the affinity of neighboring sites

35 Both positive and negative allostery

36 Motor Proteins Mediate Transport of Cargo along Cytoskeletal Filaments

37 Motor proteins convert the energy of ATP to mechanical work (power stroke)

38 Power Strokes on Myosin and Kinesin Lever arm position in ADP / free state Lever arm position in ATP / ADP P i state Similar catalytic core Vale & Milligan, Science 2000

39 Myosin: Rowers in a boat ATP hydrolysis P i release Power stroke weak actin binding ADP release ATP binding tight actin binding Two heads act independently

40 Myosin: Rowers in a boat

41 Kinesin: A Processive Step Motor ADP-ATP exchange (fast) P i & MT release (slow) ATP hydrolysis power stroke Two heads work in concert each other Vale & Milligan, Science 2000

42 Kinesin: A Processive Step Motor

43 F 1 -F 0 ATP Synthase Primary sourse of ATP for most eubacteria, eukaryotes, and archaea Reversible: functions as a ATP synthase or a proton pump Thermodynamically efficient: close to 100% MT matrix cytoplasm

44 Architecture of ATP Synthase F 1 ATPase: (a b) 3 g (de) F 0 H + pump: a b c 10

45 Rotational Catalysis in F 1 Energy not used to phosphorylate ADP, but to alter enzyme affinity for substrates and products Each ab subunit has three affinities and is used sequentially Rotary motor movement induces conf. change in each ab Boyer, Annu. Rev. Biochem. 1997

46 Rotational Catalysis in F 1 Abrahams et al, Nature 370: 621 (1994)

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48 Recommended Readings p621