INVOLVEMENT OF CONSERVED LYSINE-68 OF BACILLUS-STEAROTHERMOPHILUS LEUCINE DEHYDROGENASE IN SUBSTRATE-BINDING

Lysine 68 of Bacillus stearothermophilus leucine dehydrogenase is high ly conserved in the corresponding regions of NAD(P)(+) dependent amino acid dehydrogenase sequences. To elucidate its functional role, the l ysyl residue of the recombinant enzyme has been replaced with alanine or arginine by site directed mutagenesis. Either mutation resulted in nearly complete loss of activity in the oxidative deamination, whereas only the mutation to alanine led to a marked increase in Michaelis co nstants for both amino and keto acid substrates. On the other hand, an ionizable group in the wild-type enzyme with a pK(a) value of 10.1-10 .7, which must be protonated for binding of substrate and competitive inhibitor with an alpha-carboxyl group, was unobservable in both mutan t enzymes. These results altogether led to the conclusion that Lys 68 is located at the active site of the enzyme and involved in binding of the alpha-carboxyl group of substrate through an ionic interaction. I n addition, the alanine mutant enzyme that is almost inactive in the d eamination but significantly active in the amination was greatly stimu lated by exogenously added ammonia, suggesting that proper binding of the substrate alpha-carboxyl group at Lys-68 is essential for catalysi s.