IMPROVED SPECIFICITY TOWARD SUBSTRATES WITH POSITIVELY CHARGED SIDE-CHAINS BY SITE-DIRECTED MUTAGENESIS OF THE L-LACTATE DEHYDROGENASE OF BACILLUS-STEAROTHERMOPHILUS

The substrate specificities of L-a alpha hydroxy acid dehydrogenases, including L-lactate dehydrogenases (L-LDH's), can often be quite broad . However, an LDH with high catalytic activity toward alpha-keto acids with positively charged side chains, such as those containing ammoniu m groups, has not been described, even though there is evidence from m etabolic studies that a natural dehydrogenase with such activity might exist in Nature. L-omega-Amino-alpha-hydroxy acids are important inte rmediates in the synthesis of pharmacologically active compounds, and enzymatic reduction of omega-amino-alpha-keto acids represents an attr active route to these compounds. Graphics analysis indicated that intr oduction of acidic amino acids at position 102 of the L-LDH of Bacillu s stearothemophilus (BSLDH) would favor binding of such side chain amm onium groups. Accordingly, Q102E and Q102D mutant BSLDH's were constru cted and the steady state kinetic parameters determined for these muta nts for a broad range of a-keto acids, including omega-amino-alpha-ket o acids. The results obtained show that, compared to WT-BSLDH, these m utants show up to 25-fold improvements in k(cat)/K-m values for omega- amino-alpha-keto acid substrates.