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Stability-Activity Tradeoffs: Proximate vs. Ultimate Causes

Stability-Activity Tradeoffs: Proximate vs. Ultimate Causes. Jeffrey Endelman University of California, Santa Barbara. Causation in Biology. Proximate (physicochemical) Ultimate (evolutionary). Mayr, E. (1997) This is Biology . Cambridge: Harvard Univ. Press. LDH.

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Stability-Activity Tradeoffs: Proximate vs. Ultimate Causes

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  1. Stability-Activity Tradeoffs: Proximate vs. Ultimate Causes Jeffrey Endelman University of California, Santa Barbara

  2. Causation in Biology • Proximate (physicochemical) • Ultimate (evolutionary) Mayr, E. (1997) This is Biology. Cambridge: Harvard Univ. Press.

  3. LDH pyruvate + NADH + H+ lactate + NAD+ Enzyme Activity • Enzymes catalyze reactions, e.g. • Active site is where reaction occurs

  4. LDH pyruvate + NADH + H+ lactate + NAD+ Enzyme Activity • Enzymes catalyze reactions, e.g. • Active site is where reaction occurs • Activity measures rate of rxn • Use specific activity (per enzyme) • kcat = saturated specific activity

  5. Enzyme Stability • Enzymes denature (ND) as T inc. • DGu = GD-GN Lysozyme pH 2.5 Cp Privalov, P.L. (1979) Adv. Prot. Chem.33, 167-241. T (oC)

  6. Enzyme Stability • Enzymes denature (ND) as T inc. • DGu = GD-GN • Tm: DGu(Tm) = 0 Lysozyme pH 2.5 Cp Privalov, P.L. (1979) Adv. Prot. Chem.33, 167-241. T (oC) Tm

  7. Enzyme Stability • Enzymes denature (ND) as T inc. • DGu = GD-GN • Tm: DGu(Tm) = 0 f Creighton, T.E. (1983) Proteins. New York: Freeman. Tm T (oC)

  8. Enzyme Stability • Enzymes denature (ND) as T inc. • DGu = GD-GN • Tm: DGu(Tm) = 0 • Residual activity (Ar /Ai)

  9. Wintrode, P.L & Arnold, F.H. (2001) Adv. Prot. Chem.55, 161-225.

  10. Stability-Activity Tradeoff IPMDH 75oC 37oC 20oC Svingor, A. et al. (2001) J. Biol. Chem. 276, 28121-28125.

  11. artificial? H1: Purely Proximate IPMDH natural homologs Tradeoff exists for all enzymes.

  12. p-nitrobenzyl esterase (pNBE) Stability (Ar /Ai) Activity at 25oC (Ai) Wintrode, P.L & Arnold, F.H. (2001) Adv. Prot. Chem.55, 161-225.

  13. p-nitrobenzyl esterase (pNBE) No enzyme’s land Stability Activity at 25oC Wintrode, P.L & Arnold, F.H. (2001) Adv. Prot. Chem.55, 161-225.

  14. S/A Tradeoff Hypotheses • All enzymes have proximate tradeoff • Ultimate: Selection for high S&A Proximate: Highly optimized enzymes have S/A tradeoff

  15. Proximate Tradeoff: Flexibility • Enzymes achieve greater stability by reducing flexibility. • Flexible motions are important for catalysis in many enzymes. • Thus thermostability through reduced flexibility decreases activity. Somero, G.N. (1995) Annu. Rev. Physiol. 57, 43-68.

  16. Flexibility & Activity • Large motions (hinge bending, shear) • Pyruvate dehydrogenase • Triosephosphate isomerase • Lactate dehydrogenase • Hexokinase • Small motions (vibrational, breathing, internal rotations) • No evidence, but not unlikely Fersht, A. (1999) Structure and Mechanism in Protein Science. New York: Freeman.

  17. Proximate Tradeoff: Flexibility • Enzymes achieve greater stability by reducing flexibility. • Flexible motions are important for catalysis in many enzymes. • Thus thermostability through reduced flexibility decreases activity. Somero, G.N. (1995) Annu. Rev. Physiol. 57, 43-68.

  18. Flexibility & Stability • Stabilization involves all levels of protein structure • Experiments typically probe small motions via amide hydrogen exchange • Some thermophiles are more rigid than mesophile, others are not • “... hypothesis [that] enhanced thermal stability … [is] the result of enhanced conformational ridigity…. has no general validity.” Jaenicke, R. (2000) PNAS97, 2962-2964.

  19. Proximate Tradeoff: Flexibility • Enzymes achieve greater stability by reducing flexibility. • Flexible motions are important for catalysis in many enzymes. • Thus thermostability through reduced flexibility decreases activity. Somero, G.N. (1995) Annu. Rev. Physiol. 57, 43-68.

  20. Flexibility is Weak Link • Protein flexibility is complex • Spans picoseconds to milliseconds • Varies spatially • Only meaningful to discuss particular motions and how they affect stability and activity • Stability and activity often involve different regions and different time scales Lazaridis, T., Lee, I. & Karplus, M. (1997) Prot. Sci.6, 2589-2605.

  21. S/A Tradeoff Hypotheses • All enzymes have proximate tradeoff • Ultimate: Selection for high S&A Proximate: Highly optimized enzymes have S/A tradeoff • No known generic mechanism, e.g. flexibility • Experiments do not support notion

  22. p-nitrobenzyl esterase (pNBE) No enzyme’s land Stability Activity at 25oC

  23. Most mutations are deleterious or nearly neutral. Stability Activity at 25oC

  24. p = O(e) Mutations that improve either property are rare. Stability p = O(e) Activity at 25oC

  25. p = O(e2) Mutations that improve both properties are very rare Stability Activity at 25oC

  26. Consistent with p(S, A) = p(S) p(A) p(S>WT) = p(A>WT) = O(e) << 1 p = O(e) p = O(e2) Stability p = O(e) Activity at 25oC

  27. Proteins in nature are well-adapted: S&A are far above average frequency WT S/A

  28. Buffering/Evolvability • More mutations are nearly neutral than might be expected for random tinkering of complex system • Compartmentalization • protein domains • Redundancy • Hydrophobicity • Steric requirements Gerhart, J. & Kirschner, M. (1997) Cells, Embryos, & Evolution. Malden: Blackwell Science.

  29. Consistent with p(S, A) = p(S) p(A) p(S>WT) = p(A>WT) = O(e) << 1 p = O(e) p = O(e2) Stability p = O(e) Activity at 25oC

  30. 1 Activity (mmol/min/mg) 5 2 1 2 pNBE Melting T (oC) Directed Evolution: Improved S&A Giver, L. et al. (1998) PNAS95, 12809-12813.

  31. S/A Tradeoff Hypotheses • All enzymes have proximate tradeoff • Ultimate: Selection for high S&A Proximate: Highly optimized enzymes have S/A tradeoff • Proximate: Most mutations are deleterious or nearly neutral Ultimate: Selection for threshold S&A Wintrode, P.L & Arnold, F.H. (2001) Adv. Prot. Chem.55, 161-225.

  32. H3: Mutation-Selection Lethal Viable Stability Activity at 25oC

  33. Threshold Selection • DGu(Th) = c kTh • KD/N = e-c • Proteins typically have c > 7 • No reason (or evidence) to believe higher S has selective advantage

  34. Threshold Selection • DGu(Th) = c kTh • KD/N = e-c • Proteins typically have c > 5 • No reason (or evidence) to believe higher S has selective advantage • A(Th) = a • With low flux control coefficient, higher A may offer no advantage • When important for control, higher A may be disadvantageous

  35. H3: Mutation-Selection Lethal Viable Stability Activity at 25oC

  36. Mutation brings S&A to thresholds Lethal Viable Stability Activity at 25oC

  37. DGu(Th) kTh S/A for H3 (Mutation-Selection) 20oC c 75oC 37oC a A(Th)

  38. S/A in Nature IPMDH 75oC 37oC 20oC = A(To) Svingor, A. et al. (2001) J. Biol. Chem. 276, 28121-28125.

  39. melting Arrhenius A a T Th

  40. T 75oC Th 37oC 20oC a A

  41. T 75oC To 37oC 20oC a A

  42. DGu(Th) kTh S/A for H3 (Mutation-Selection) 75oC c 37oC 20oC a A(To)

  43. 37oC 20oC 75oC Th Tm T DGu/kT c 0

  44. 37oC 20oC 75oC T DGu/kT c 0

  45. 37oC 20oC 75oC Tm Tm Tm DGu/kT 0

  46. S/A for H3 (Mutation-Selection) 75oC 37oC Tm 20oC a A(To)

  47. S/A in Nature IPMDH 75oC 37oC 20oC Svingor, A. et al. (2001) J. Biol. Chem. 276, 28121-28125.

  48. Conclusions • Because biological phenotypes are well-adapted, most mutations are deleterious • This mutational pressure pushes phenotypes to the thresholds of selection • Selection that requires homologs to have comparable S&A at physiological temperatures creates the appearance of S/A tradeoffs at a reference temperature • The proximate causes for S&A among homologs are unlikely to be universal

  49. Performance Tradeoffs • Pervasive in biological thinking • Resource allocation (time, energy, mass) • Design tradeoffs • Biochemistry: Stability/Activity • Behavior: Foraging, Fight/Flight • Physiology: Respiration, Biomechanics

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