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Title
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Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole.
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Authors
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D.A.Kraut,
P.A.Sigala,
B.Pybus,
C.W.Liu,
D.Ringe,
G.A.Petsko,
D.Herschlag.
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Ref.
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Plos Biol, 2006,
4,
e99-519.
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PubMed id
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Abstract
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A longstanding proposal in enzymology is that enzymes are electrostatically and
geometrically complementary to the transition states of the reactions they
catalyze and that this complementarity contributes to catalysis. Experimental
evaluation of this contribution, however, has been difficult. We have
systematically dissected the potential contribution to catalysis from
electrostatic complementarity in ketosteroid isomerase. Phenolates, analogs of
the transition state and reaction intermediate, bind and accept two hydrogen
bonds in an active site oxyanion hole. The binding of substituted phenolates of
constant molecular shape but increasing pK(a) models the charge accumulation in
the oxyanion hole during the enzymatic reaction. As charge localization
increases, the NMR chemical shifts of protons involved in oxyanion hole hydrogen
bonds increase by 0.50-0.76 ppm/pK(a) unit, suggesting a bond shortening of 0.02
A/pK(a) unit. Nevertheless, there is little change in binding affinity across a
series of substituted phenolates (DeltaDeltaG = -0.2 kcal/mol/pK(a) unit). The
small effect of increased charge localization on affinity occurs despite the
shortening of the hydrogen bonds and a large favorable change in binding
enthalpy (DeltaDeltaH = -2.0 kcal/mol/pK(a) unit). This shallow dependence of
binding affinity suggests that electrostatic complementarity in the oxyanion
hole makes at most a modest contribution to catalysis of 300-fold. We propose
that geometrical complementarity between the oxyanion hole hydrogen-bond donors
and the transition state oxyanion provides a significant catalytic contribution,
and suggest that KSI, like other enzymes, achieves its catalytic prowess through
a combination of modest contributions from several mechanisms rather than from a
single dominant contribution.
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