The catalytic mechanism for the interconversion of pyruvate to L-lactate by the enzyme L-lactate dehydrogenase (LDH), in the presence of the cofactor nicotinamide adenine dinucleotide (NAD), has been studied using semiempirical AM1 quantum mechanical calculations. We have characterized the structure of the LDH transition state (TS), in isolation and in the presence of key active-site groups, using a supermolecule model. An initial investigation with isolated substrate and cofactor analogues resulted in TS structures for hydride-ion transfer from the cofactor analogue, planar trans-1- methyldihydronicotinamide to eight conformers of the substrate analogue, protonated pyruvic acid. Fragments of essential active-site residues were then introduced in stages. With truncated Arg-171 and His-195 residues, the TS for hydride transfer from the cofactor analogue to the substrate pyruvate resembled the active-site configuration in the X-ray crystallographic structure of the abortive LDH-NADH-oxamate ternary complex. The substrate species is carbonyl-protonated and thus the rate-limiting chemical step is hydride transfer. These results contrast with earlier work indicating that carbonyl-protonated pyruvate is unstable in the free state (K. E. Norris, G. B. Bacskay and J. E. Gready, J. Comput. Chem., 1993, 14, 699). Introduction of the Val-138 fragment gave closer agreement with experiment for the orientation of the cofactor analogue's carboxamide side chain in the TS and for the reversibility criteria for the reaction. For each TS located, stable reactant and product complexes have been isolated by following the reaction coordinate, and the optimized structures, energies and charge distributions of the TS, stable reactant and product complexes and the isolated reactants and products are reported. There is significant charge transfer in the TS, with a charge of ca. +0.4 on the nicotinamide species.
|Number of pages||10|
|Journal||Journal of the Chemical Society, Faraday Transactions|
|Publication status||Published - 1994|