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 deh ydrogenase from Bacillus stearothermophilus has been mutated into Ala, Arg, or Gln. All of the mutant enzymes had markedly reduced activitie s in the oxidative deamination, whereas the Michaelis constants for su bstrate and coenzyme did not change significantly upon the mutation, e xcept for a 10-30-fold increase in K(m) values for alpha-keto-iso-capr oate 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 pK(a) values of 8.9 and 10.7 must be u nprotonated for catalysis and protonated for substrate binding, respec tively. Combined with the analyses of solvent isotope effect and inhib ition by substrate analogs, these results unequivocally show that the epsilon-amino group of Lys-80 participates in catalysis as a general b ase, assisting the nucleophilic attack of a water molecule to the subs trate alpha-carbon atom. Furthermore, the Ala mutant was markedly stim ulated by primary amines depending on the pK(a) and molecular volume, suggesting that in the Ala mutant the added amines can partially repla ce the general base function of Lys-80 in the wild type enzyme.