Stability-Activity Tradeoffs: Proximate vs. Ultimate Causes

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
Enzyme Activity

• Enzymes catalyze reactions, e.g.
• Active site is where reaction occurs

4
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
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 enzymes 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 SA
• 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.
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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) PNAS 97, 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.
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S/A Tradeoff Hypotheses

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

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p-nitrobenzyl esterase (pNBE)
No enzymes land
Stability
Activity at 25oC
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Most mutations are deleterious or nearly neutral.
Stability
Activity at 25oC
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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
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Consistent with p(S, A) p(S) p(A) p(SgtWT)
p(AgtWT) O(e) ltlt 1
p O(e2)
p O(e)
Stability
p O(e)
Activity at 25oC
27

• Proteins in nature are well-adapted
• SA are far above average

frequency
WT
S/A
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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(SgtWT)
p(AgtWT) O(e) ltlt 1
p O(e2)
p O(e)
Stability
p O(e)
Activity at 25oC
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Directed Evolution Improved SA
Giver, L. et al. (1998) PNAS 95, 12809-12813.
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S/A Tradeoff Hypotheses

• All enzymes have proximate tradeoff
• Ultimate Selection for high SA
• Proximate Highly optimized enzymes have S/A
  tradeoff
• Proximate Most mutations are deleterious or
  nearly neutral
• Ultimate Selection for threshold SA

Wintrode, P.L Arnold, F.H. (2001) Adv. Prot.
Chem. 55, 161-225.
32
H3 Mutation-Selection
Viable
Lethal
Stability
Activity at 25oC
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Threshold Selection

• DGu(Th) c kTh
• KD/N e-c
• Proteins typically have c gt 7
• No reason (or evidence) to believe higher S has
  selective advantage

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Threshold Selection

• DGu(Th) c kTh
• KD/N e-c
• Proteins typically have c gt 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

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H3 Mutation-Selection
Viable
Lethal
Stability
Activity at 25oC
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Mutation brings SA to thresholds
Viable
Lethal
Stability
Activity at 25oC
37
S/A for H3 (Mutation-Selection)
20oC
c
75oC
37oC
a
A(Th)
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S/A in Nature
IPMDH
75oC
37oC
20oC
A(To)
Svingor, A. et al. (2001) J. Biol. Chem. 276,
28121-28125.
39
A
a
T
Th
40
T
75oC
37oC
Th
20oC
a
A
41
T
75oC
37oC
To
20oC
a
A
42
S/A for H3 (Mutation-Selection)
75oC
c
37oC
20oC
a
A(To)
43
37oC
20oC
75oC
T
Th
Tm
DGu/kT
c
0
44
37oC
20oC
75oC
T
DGu/kT
c
0
45
37oC
20oC
75oC
Tm
Tm
Tm
DGu/kT
0
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S/A for H3 (Mutation-Selection)
75oC
37oC
Tm
20oC
a
A(To)
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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 SA at physiological temperatures
  creates the appearance of S/A tradeoffs at a
  reference temperature
• The proximate causes for SA among homologs are
  unlikely to be universal

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Performance Tradeoffs

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