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Mechanistic Analysis of Beta‐Lactam Synthetase and the Influence of Conformational Fluctuations
Author(s) -
Raber Mary L,
Townsend Craig A.
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.1_supplement.611.6
Subject(s) - chemistry , lactam , arrhenius plot , kinetic isotope effect , viscosity , reaction rate constant , arrhenius equation , conformational change , conformational isomerism , stereochemistry , kinetics , crystallography , thermodynamics , activation energy , organic chemistry , molecule , deuterium , physics , quantum mechanics
β‐lactam synthetase (β‐LS) catalyzes formation of the key β‐lactam ring of the potent β‐lactamase inhibitor, clavulanic acid, in an ATP/Mg 2+ dependent reaction. In the present study, β‐LS was subjected to pH, pD, temperature, and viscosity variation. The k cat of β‐LS changes very little with varying pH, while its second‐order rate constant can be fitted to three ionizable groups. A normal SIE on k cat of 1.44 ± 0.17 was found, while an inverse solvent isotope effect of 0.80 ± 0.07 was determined for k cat / K m . A relative viscosity versus reciprocal rate plot of k cat gave a slope of around two. This suggests that a conformational change is rate‐limiting, with the small SIE from the increased viscosity of D 2 O relative to H 2 O. The k cat Arrhenius plots are linear and the corresponding parameters represent the enthalpically controlled energetics of the conformational change. In contrast, k cat / K m is significantly enhanced with increasing viscosity, and Arrhenius plots of k cat / K m and K m were non‐linear. From the analyses presented, it is proposed that an equilibrium of enzyme conformers exists before the first irreversible step in the β‐LS catalytic mechanism to match one later in the reaction coordinate corresponding to product release. This research was supported by NIH grant RO1 AI014937.