| Hydrogen tunneling has been observed in a substrate binding site mutant (F93W) of horse liver alcohol dehydrogenase (LADH) and has been shown to be modulated by the size of a single side chain (Val203) that is distal to the nicotinamide ring of the NAD+ cofactor. To investigate the role of other residues in the tunneling process, twenty-six double or triple mutants of F93W have been constructed and characterized. Mutation of residues that line the cofactor binding site, but are removed from the nicotinamide ring, were generally found to affect the binding of cofactor with very little effect on catalytic efficiency and tunneling. Mutation of highly conserved residues that are very remote from the active site also had little effect on catalysis. In contrast, mutation of a residue in the nicotinamide binding site was found to greatly decrease the catalytic efficiency of the enzyme without decreasing the apparent tunneling contribution to catalysis.; Overall, the results of mutations made in LADH suggest a role for proximal, but not remote side chains in the modulation of hydrogen tunneling in the enzyme. Furthermore, the results of the present studies suggest that there is a separation of binding and catalysis in the enzyme, such that a particular residue could affect just one or both of these steps. For mutants where binding or catalytic efficiency is affected in the absence of an effect on hydrogen tunneling, it is proposed that there is a step prior to catalysis, wherein the enzyme must attain a particular configuration. In this context, mutation of a residue could affect the ability of the enzyme to reach the reactive configuration. However, once the configuration is attained, the system would undergo the reaction with the associated hydrogen tunneling, unaffected by the mutation. |
