Involvement of conserved lysine 68 of Bacillus stearothermophilus leucine dehydrogenase in substrate binding.
Lysine 68 of Bacillus stearothermophilus leucine dehydrogenase is highly conserved in the corresponding regions of NAD(P)+-dependent amino acid dehydrogenase sequences. To elucidate its functional role, the lysyl 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 constants for both amino and keto acid substrates. On the other hand, an ionizable group in the wild-type enzyme with a pKa 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 mutant 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. In addition, the alanine mutant enzyme that is almost inactive in the deamination but significantly active in the amination was greatly stimulated by exogenously added ammonia, suggesting that proper binding of the substrate alpha-carboxyl group at Lys-68 is essential for catalysis.
Sekimoto
T
T
Institute of Scientific and Industrial Research, Osaka University, Japan.
Fukui
T
T
Tanizawa
K
K
D000409:Alanine / Q000378:metabolism; D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000641:Ammonia / Q000494:pharmacology; D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D050803:Leucine Dehydrogenase; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D000641:Ammonia; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase; D008239:Lysine; D000409:Alanine