Active site hydrophobic residues impact hydrogen tunneling differently in a thermophilic alcohol dehydrogenase at optimal versus nonoptimal temperatures.

Biochemistry
Zachary D NagelJudith P Klinman

Abstract

A growing body of data suggests that protein motion plays an important role in enzyme catalysis. Two highly conserved hydrophobic active site residues in the cofactor-binding pocket of ht-ADH (Leu176 and V260) have been mutated to a series of hydrophobic side chains of smaller size, as well as one deletion mutant, L176Δ. Mutations decrease k(cat) and increase K(M)(NAD(+)). Most of the observed decreases in effects on k(cat) at pH 7.0 are due to an upward shift in the optimal pH for catalysis; a simple electrostatic model is invoked that relates the change in pK(a) to the distance between the positively charged nicotinamide ring and bound substrate. Structural modeling of the L176Δ and V260A variants indicates the development of a cavity behind the nicotinamide ring without any significant perturbation of the secondary structure of the enzyme relative to that of the wild type. Primary kinetic isotope effects (KIEs) are modestly increased for all mutants. Above the dynamical transition at 30 °C for ht-ADH [Kohen, A., et al. (1999) Nature 399, 496], the temperature dependence of the KIE is seen to increase with a decrease in side chain volume at positions 176 and 260. Additionally, the relative trends in the temperature dependence...Continue Reading

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Citations

Jan 10, 2013·Journal of Biomolecular Structure & Dynamics·Teodorico C RamalhoElaine F F da Cunha
Oct 29, 2013·Archives of Biochemistry and Biophysics·Daniel RostonAmnon Kohen
Jun 12, 2013·Annual Review of Biochemistry·Judith P Klinman, Amnon Kohen
May 16, 2013·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Daniel RostonAmnon Kohen
Dec 30, 2014·Accounts of Chemical Research·Judith P Klinman
Aug 27, 2014·Biochemistry·Savarimuthu Baskar RajBryce V Plapp
Jun 24, 2014·Biochimica Et Biophysica Acta·Ana Sánchez-AzquetaMilagros Medina
Jan 31, 2018·Protein Science : a Publication of the Protein Society·David D BoehrKathleen F O'Rourke
Sep 12, 2014·The Journal of Biological Chemistry·Judith P Klinman, Amnon Kohen
Mar 26, 2013·The Journal of Biological Chemistry·Zachary D NagelJudith P Klinman
Jun 22, 2019·Annual Review of Biochemistry·Judith P Klinman
May 7, 2020·Proceedings of the National Academy of Sciences of the United States of America·Jianyu ZhangJudith P Klinman
Aug 24, 2021·Journal of Biomolecular Structure & Dynamics·Kristen Rhinehardt, Ming Dong

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