CATALYTIC-RATE IMPROVEMENT OF A THERMOSTABLE MALATE-DEHYDROGENASE BY A SUBTLE ALTERATION IN COFACTOR BINDING

The nucleotide-binding fold of many NAD(+)-dependent dehydrogenases contains a conserved acidic amino acid residue which hydrogen-bonds with the 2'- and 3'-hydroxy groups of the adenine-ribose of the cofactor. This residue is highly conserved as aspartate in malate dehydrogenases, except in the thermophilic enzyme from Termus aquaticus B (TaqMDH), which has glutamic acid-41 in the equivalent position. The catalytic mechanism was dissected to investigate the functional significance of this difference in TagMDH with respect to a mutant enzyme where glutamic acid-41 was replaced by aspartic acid. The mutant enzyme was found to retain a high degree of protein structural stability to both thermal and chemical denaturation. When compared with the wild-type enzyme the mutant had a higher K-m and K-d for both reduced and oxidized cofactors (NADH and NAD(+)) and a 2-3-fold increase in steady-state k(cat.) in both assay directions. The rate-determining step for the reduction of oxaloacetate by wild-type TaqMDH was shown to be the rate of NAD(+) release, which was about 2.5-fold higher for the mutant enzyme. This correlates well with the 1.8-fold higher steady-state k(cat.) of the mutant enzyme and represents an improvement in the steady-state k(cat.) of a thermophilic enzyme at moderate temperature by a conservative amino acid substitution which increases the rate of product release.