BIOL 181 |
| 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 bacterial 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 oxyanion geometry but varying pKa models the charge accumulation in the oxyanion hole during the enzymatic reaction. As charge localization increases, the NMR chemical shifts of protons in oxyanion hole hydrogen bonds increase by ~0.50 ppm/pKa unit, suggesting a bond shortening of ~0.02 Å/pKa unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates. 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. This shallow dependence suggests that electrostatic complementarity in the oxyanion hole and, more broadly, in the active site environment plays, at most, a modest role in catalysis. Geometrical complementarity between the oxyanion hydrogen bond donors and the transition state oxyanion may provide a significant contribution to catalysis, and we suggest that KSI, like other enzymes, achieves its catalytic prowess through a combination of modest contributions from several mechanisms rather than from one or two dominant contributions. |
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Repligen Award Symposium: Enzymatic Catalysis and Transition States
1:30 PM-4:40 PM, Wednesday, 13 September 2006 Moscone Center -- Room 238, Oral
Division of Biological Chemistry |