Testing Electrostatic Complementarity in Enzyme Catalysis: Hydrogen Bonding in the Ketosteroid Isomerase Oxyanion Hole

Abstract

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 p K 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/p K a unit, suggesting a bond shortening of ˜0.02 Å/p K a unit. Nevertheless, there is little change in binding affinity across a series of substituted phenolates (ΔΔG = −0.2 kcal/mol/p K 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 (ΔΔH = −2.0 kcal/mol/p K 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.

Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase

“…The k cat /K M value for S mini of 20 Ϯ 2 M Ϫ1 s Ϫ1 is Ϸ10 4 -fold lower than the value for S full . Similarly, the binding affinity for a single-ring phenolate transition state analog is reduced Ϸ10 6 -fold relative to the binding affinity for a multiple-ringed steroid transition state analog (equilenin) (52). This large decrease in k cat /K M for S mini versus S full and in the binding affinity for a single-ring versus a multiple-ring transition state analog is consistent with a loss in binding interactions with the distal steroid rings (40,41,52).…”

“…8 of the same ref.). The similar catalysis of bound single-and multiple-ringed substrates stands in contrast to the 6 kcal/mol or 10 4 -fold predicted difference in catalysis and supports the use of phenolates as an incisive probe of electrostatics in the KSI oxyanion hole (52). Nevertheless, additional tests are desirable (e.g., 55) and contributions from oxyanion hole geometric changes and electrostatic interactions at the site of general base catalysis remain to be dissected (54,56).…”

“…For KSI, interactions with the remote steroid rings help localize the substrate to the active site and interactions with the proximal rings position the substrate with respect to the active site general base and oxyanion hole hydrogen bonds. These hydrogen bonds appear to be geometrically optimized for transition state interactions, and there may also be a modest contribution from preferential electrostatic interactions within the oxyanion hole relative to aqueous solution (48,52,56,61,62).…”

“…Similarly, the binding affinity for a single-ring phenolate transition state analog is reduced Ϸ10 6 -fold relative to the binding affinity for a multiple-ringed steroid transition state analog (equilenin) (52). This large decrease in k cat /K M for S mini versus S full and in the binding affinity for a single-ring versus a multiple-ring transition state analog is consistent with a loss in binding interactions with the distal steroid rings (40,41,52). Indeed, crystal structures show a constellation of hydrophobic residues that pack around the distal steroid rings that is essentially identical for enzyme-transition state analog and enzyme-product complexes (40,41).…”

“…In a previous study, Kraut et al used a series of phenolate analogs to experimentally test the role of electrostatic complementarity in KSI's oxyanion hole, and the results suggested that oxyanion hole electrostatic complementarity provides only a modest contribution to catalysis (52). Nevertheless, it was not determined whether the behavior of single-ring phenolate transition state analogs accurately reflect the change in oxyanion hole electrostatics.…”