Transcript Enzyme Evolution: The Role of Mutation

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.
Enzyme Activity
• Enzymes catalyze reactions, e.g.
LDH
pyruvate + NADH + H+  lactate + NAD+
• Active site is where reaction occurs
Enzyme Activity
• Enzymes catalyze reactions, e.g.
LDH
pyruvate + NADH + H+  lactate + NAD+
• Active site is where reaction occurs
• Activity measures rate of rxn
– Use specific activity (per enzyme)
– kcat = saturated specific activity
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)
Enzyme Stability
• Enzymes denature (ND) as T inc.
• DGu = GD-GN
Lysozyme
• Tm: DGu(Tm) = 0
pH 2.5
Cp
Privalov, P.L. (1979) Adv. Prot. Chem.
33, 167-241.
Tm
T (oC)
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)
Enzyme Stability
•
•
•
•
Enzymes denature (ND) as T inc.
DGu = GD-GN
Tm: DGu(Tm) = 0
Residual activity (Ar /Ai)
Wintrode, P.L & Arnold, F.H. (2001) Adv. Prot. Chem. 55, 161-225.
Stability-Activity Tradeoff
90
IPMDH
melting T (oC)
85
75oC
80
75
70
37oC
65
20oC
60
55
50
0
2
4
6
8
10
kcat (s-1) at 20oC
Svingor, A. et al. (2001) J. Biol. Chem. 276, 28121-28125.
12
H1: Purely Proximate
90
IPMDH
melting T (oC)
85
80
natural homologs
75
artificial?
70
65
Tradeoff exists for all
enzymes.
60
55
50
0
2
4
6
8
kcat (s-1) at 20oC
10
12
Stability (Ar /Ai)
p-nitrobenzyl esterase (pNBE)
Activity at 25oC (Ai)
Wintrode, P.L & Arnold, F.H. (2001) Adv. Prot. Chem. 55, 161-225.
Stability
p-nitrobenzyl esterase (pNBE)
No enzyme’s land
Activity at 25oC
Wintrode, P.L & Arnold, F.H. (2001) Adv. Prot. Chem. 55, 161-225.
S/A Tradeoff Hypotheses
1. All enzymes have proximate tradeoff
2. Ultimate: Selection for high S&A
Proximate: Highly optimized enzymes
have S/A tradeoff
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.
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.
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.
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.
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.
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.
S/A Tradeoff Hypotheses
1. All enzymes have proximate tradeoff
2. 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
Stability
p-nitrobenzyl esterase (pNBE)
No enzyme’s land
Activity at 25oC
Stability
Most mutations
are deleterious or
nearly neutral.
Activity at 25oC
Stability
p = O(e)
Mutations that
improve either
property are rare.
p = O(e)
Activity at 25oC
Stability
p = O(e2)
Mutations that
improve both
properties are very
rare
Activity at 25oC
Consistent with p(S, A) = p(S) p(A)
p(S>WT) = p(A>WT) = O(e) << 1
p = O(e2)
Stability
p = O(e)
p = O(e)
Activity at 25oC
Proteins in nature are well-adapted:
S&A are far above average
frequency
WT
S/A
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.
Consistent with p(S, A) = p(S) p(A)
p(S>WT) = p(A>WT) = O(e) << 1
p = O(e2)
Stability
p = O(e)
p = O(e)
Activity at 25oC
Activity (mmol/min/mg)
Directed Evolution: Improved S&A
1
5
2
1
2
pNBE
Melting T (oC)
Giver, L. et al. (1998) PNAS 95, 12809-12813.
S/A Tradeoff Hypotheses
1. All enzymes have proximate tradeoff
2. Ultimate: Selection for high S&A
Proximate: Highly optimized enzymes
have S/A tradeoff
3. 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.
Stability
H3: Mutation-Selection
Lethal
Activity at 25oC
Viable
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
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
Stability
H3: Mutation-Selection
Lethal
Activity at 25oC
Viable
Stability
Mutation brings S&A to thresholds
Lethal
Activity at 25oC
Viable
S/A for H3 (Mutation-Selection)
DGu(Th)
kTh
c
20oC
37oC
75oC
a
A(Th)
S/A in Nature
90
melting T (oC)
85
IPMDH
75oC
80
75
70
37oC
65
60
20oC
55
50
0
2
4
6
8
10
kcat (s-1) at 20oC = A(To)
Svingor, A. et al. (2001) J. Biol. Chem. 276, 28121-28125.
12
A
a
melting
Arrhenius
Th
T
T
75oC
Th 37oC
20oC
a
A
T
75oC
To 37oC
20oC
a
A
S/A for H3 (Mutation-Selection)
DGu(Th)
kTh
75oC
37oC
c
20oC
a
A(To)
DGu/kT
c
0
20oC
37oC 75oC
Th
Tm
T
DGu/kT
c
0
20oC
37oC 75oC
T
DGu/kT
0
20oC
37oC 75oC
Tm Tm Tm
S/A for H3 (Mutation-Selection)
75oC
Tm
37oC
20oC
a
A(To)
S/A in Nature
90
melting T (oC)
85
IPMDH
75oC
80
75
70
37oC
65
60
20oC
55
50
0
2
4
6
8
10
kcat (s-1) at 20oC
Svingor, A. et al. (2001) J. Biol. Chem. 276, 28121-28125.
12
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
Performance Tradeoffs
•
•
•
•
•
•
Pervasive in biological thinking
Resource allocation (time, energy, mass)
Design tradeoffs
Biochemistry: Stability/Activity
Behavior: Foraging, Fight/Flight
Physiology: Respiration, Biomechanics