Active Site Hydrophobic Residues Impact Hydrogen Tunneling Differently in a Thermophilic Alcohol Dehydrogenase at Optimal vs. Non-Optimal Temperatures

摘要

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 kcat and increase KM(NAD⁺). Most of the observed decreases in effects on kcat 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 pKa 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 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 et al., Nature (1999) 399, 496), the temperature dependence of the KIE is seen to increase with decreasing side chain volume at positions 176 and 260. Additionally, the relative trends in the temperature dependence of the KIE above and below 30 °C appear reversed for the cofactor-binding pocket mutants in relation to wild-type protein. The aggregate results are interpreted in the context of a full tunneling model of enzymatic hydride transfer that incorporates both protein conformational sampling (preorganization) and active site optimization of tunneling (reorganization). The reduced temperature dependence of the KIE in the mutants below 30 °C indicates that at low temperature, the enzyme adopts conformations refractory to donor-acceptor distance sampling.