Detection of large pKa perturbations of an inhibitor and a catalytic group at an enzyme active site, a mechanistic basis for catalytic power of many enzymes

Nam Chul Ha, Min Sung Kim, Weontae Lee, Kwan Yong Choi, Byung Ha Oh

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Abstract

Δ5-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pKa ∼16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr14 (pKa ∼11.5), and the protonation of the catalytic residue Asp38 with pKa of ∼4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pKa values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pKa difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pKa difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pKa perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pKa value as high as ∼30.

Original languageEnglish
Pages (from-to)41100-41106
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number52
DOIs
Publication statusPublished - 2000 Dec 29

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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