Evidence for lysine 80 as general base catalyst of leucine dehydrogenase.
To elucidate the functional role of the lysyl residue highly conserved in NAD(P)(+)-dependent amino acid dehydrogenases, Lys-80 of leucine dehydrogenase from Bacillus stearothermophilus has been mutated into Ala, Arg, or Gln. All of the mutant enzymes had markedly reduced activities in the oxidative deamination, whereas the Michaelis constants for substrate and coenzyme did not change significantly upon the mutation, except for a 10-30-fold increase in Km values for alpha-keto-iso-caproate in the Ala and Gln mutants. The pH profiles of kinetic parameters of the mutants considerably differed from those of the wild type, in which two ionizable groups with pKa values of 8.9 and 10.7 must be unprotonated for catalysis and protonated for substrate binding, respectively. Combined with the analyses of solvent isotope effect and inhibition by substrate analogs, these results unequivocally show that the epsilon-amino group of Lys-80 participates in catalysis as a general base, assisting the nucleophilic attack of a water molecule to the substrate alpha-carbon atom. Furthermore, the Ala mutant was markedly stimulated by primary amines depending on the pKa and molecular volume, suggesting that in the Ala mutant the added amines can partially replace the general base function of Lys-80 in the wild type enzyme.
Sekimoto
T
T
Institute of Scientific and Industrial Research, Osaka University, Japan.
Matsuyama
T
T
Fukui
T
T
Tanizawa
K
K
D000588:Amines / Q000494:pharmacology; D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004789:Enzyme Activation; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007651:Keto Acids / Q000494:pharmacology; D050803:Leucine Dehydrogenase; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation
D000588:Amines; D017931:DNA Primers; D007651:Keto Acids; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase; D008239:Lysine