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  • A critical test of the "tunneling and coupled motion" concept in enzymatic alcohol oxidation.

A critical test of the "tunneling and coupled motion" concept in enzymatic alcohol oxidation.

Journal of the American Chemical Society (2013-09-12)
Daniel Roston, Amnon Kohen
ABSTRACT

The physical mechanism of C-H bond activation by enzymes is the subject of intense study, and we have tested the predictions of two competing models for C-H activation in the context of alcohol dehydrogenase. The kinetic isotope effects (KIEs) in this enzyme have previously suggested a model of quantum mechanical tunneling and coupled motion of primary (1°) and secondary (2°) hydrogens. Here we measure the 2° H/T KIEs with both H and D at the 1° position and find that the 2° KIE is significantly deflated with D-transfer, consistent with the predictions of recent Marcus-like models of H-transfer. The results suggest that the fast dynamics of H-tunneling result in a 1° isotope effect on the structure of the tunneling ready state: the trajectory of D-transfer goes through a shorter donor-acceptor distance than that of H-transfer.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Alcohol Dehydrogenase from Saccharomyces cerevisiae, powder, ≥300 units/mg protein, mol wt ~141,000 (four subunits)
Sigma-Aldrich
Alcohol Dehydrogenase from Saccharomyces cerevisiae
Sigma-Aldrich
Alcohol Dehydrogenase from Saccharomyces cerevisiae, ≥300 units/mg protein, lyophilized powder (contains buffer salts), Mw 141-151 kDa
Sigma-Aldrich
Alcohol Dehydrogenase equine, recombinant, expressed in E. coli, ≥0.5 U/mg