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β‐Secondary deuterium isotope effect and solvent isotope effects in catalysis by subtilisin BPN′
Author(s) -
Kovach Ildiko M.,
Do Son,
Schowen Richard L.
Publication year - 1990
Publication title -
journal of physical organic chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.325
H-Index - 66
eISSN - 1099-1395
pISSN - 0894-3230
DOI - 10.1002/poc.610030409
Subject(s) - chemistry , kinetic isotope effect , deuterium , tetrahedral carbonyl addition compound , reaction rate constant , catalysis , stereochemistry , nucleophile , crystallography , medicinal chemistry , kinetics , organic chemistry , physics , quantum mechanics
Abstract Subtilisin BPN′ catalyzes the hydrolysis in protium and deuterium oxides of p ‐NO 2 C 6 H 4 OCOCL 2 NHCO 2 CH 2 C 6 H 5 (L = H, D) in the pH(D) range 5·0–8·5 (H 2 O) and 5·4–9·0 (D 2 O), according to simple Michaelis–Menten kinetics. The parameter k cat / K m exhibits pH(D) inflection points of 7·17 ± 0·05 (H 2 O) and 7·69 ± 0·08 (D 2 O), and k cat shows 6·88 ± 0·05 (H 2 O) and 7·50 ± 0·07 (D 2 O). The ‘normal’ Δp K values of 0·5–0·6 indicate no unusual effects of D 2 O on enzyme properties. The solvent isotope effects (H 2 O/D 2 O) on the limiting values of the rate constants at high pH(D) are 1·13 ± 0·07 for k cat / K m and 1·29 ± 0·05 for K cat . These small effects indicate no more than minor contributions of general acid–base catalysis for rate‐limiting events for either k cat / K m or k cat . The β‐deuterium secondary isotope effects (2H/2D) are roughly estimated by extrapolation as 0·95 ± 0·01 for k cat / K m , corresponding to substantial tetrahedral character in the transition state, and 1·03 ± 0·03 for k cat , consistent with no tetrahedral character. Models consistent with these results have as rate‐limiting events for k cat / K m nucleophilic attack by active‐site imidazole and for k cat , among other possibilities, the release of carboxylate product from the imidazolium form of the enzyme.