Catalytic role of enzymes: Short strong H-bond-induced partial proton shuttles and charge redistributions

Abstract

A two-step reaction mechanism (catalyzed alternatively by acid and base) with partial proton shuttles and charge redistributions promoted by short strong H bonds (SSHBs) (playing a dual role as an amphi-acid͞base catalyst) is proposed to explain the enormous rate enhancement observed in enzymatic reactions involving carbanion intermediates. The SSHBs in the two-step reactions are found to be responsible for enhancing enzymesubstrate interactions in favor of the transition state structure over that of reactant. The detailed quantum theoretical studies of ketosteroid isomerase provide evidence of assisting roles of SSHB in enzymatic activity. The understanding of the two-step reaction mechanism would be a useful aid in designing novel functional enzymes and abzymes.A n understanding of how enzymes enhance the rate of reactions is essential for investigating the biological role of enzymes and designing new enzymes (1-3). In this context, enzyme-substrate interactions and enzyme preorganization have been invoked to explain the rate enhancement in favor of substrate transition state (TS) structures (4-14). In enzymesubstrate interactions, low barrier H bonds characterized by short and strong H bonds (SSHB) have been considered responsible for drastic enhancement in H-bond energies (4)(5)(6)(7)(8)(9)(10)(11)15). This concept has been introduced to explain the fast reactivities observed in various enzymes (4). Alternative explanations like preorganization in favor of the TS structures (mainly by electrostatic interactions) have also been proposed (12, 13), and the issue has been highly debated (9-24). In the present study, we attempt to resolve this issue by elucidating the origin of the catalytic role in ketosteroid isomerase (KSI), which is representative of this class of enzymes. We have carried out quantum theoretical calculations on the active sites of KSI to compare the experimental x-ray structures, NMR chemical shifts, and kinetic reaction rates for various mutants. We find evidence that the SSHB [driven by preorganized reaction environment in favor of the TS structure over the reactant structure, or enzymesubstrate complex (ES)] promotes both partial proton shuttles and (electronic) charge redistributions in a two-step mechanism as the role of an amphi-acid͞base catalyst, and hence eventually leads to a drastic lowering of the activation barrier in the catalytic reaction.KSI is one of the most proficient enzymes, catalyzing the isomerization of a variety of ⌬ 5 -3-ketosteroids to ⌬ 4 -3-ketosteroids (i.e., promoting an allylic rearrangement involving intramolecular proton transfer via a dienolic intermediate) (Fig. 1), with diffusion-controlled reactivity, and serves as a paradigm for fast enzymatic enolization involving carbanions (25-35). Enzyme reactions associated with carbanion intermediates responsible for isomerization reaction and carbon-carbon bond formation͞cleavage are vital to metabolism of living organisms.The important catalytic residues in Pseudomonas testosteroni KSI (TI) and Pseudomonas putid...

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