ROLE OF THE CONSERVED GLYCYL RESIDUES LOCATED AT THE ACTIVE-SITE OF LEUCINE DEHYDROGENASE FROM BACILLUS-STEAROTHERMOPHILUS

A tetrapeptide sequence, Gly-Gly-(Gly/Ala)-Lys, containing a catalytic ally important lysyl residue, is highly conserved in NAD(P)(+)-depende nt amino acid dehydrogenases. To elucidate functional roles of the gly cyl residues in this conserved sequence, Gly-77, Gly-78, and Gly-79 of the recombinant leucine dehydrogenase from Bacillus stearothermophilu s have been individually replaced with Ala by site-directed mutagenesi s. All of the mutant enzymes had Michaelis constants for alpha-keto-is o-caproate and ammonia several times larger than the wild-type enzyme while retaining considerable catalytic activities. However, inhibition constants for a substrate analog without an alpha-carbonyl group were unchanged by the mutations. On the other hand, the rate of inactivati on by pyridoxal 5'-phosphate and the microenvironment of aromatic resi dues, in particular of the sole tryptophanyl residue (Trp-46) located in the vicinity of the active site, were affected by the mutations of the glycyl residues. All of these results suggest that the conserved g lycyl residues are important for fine-tuning of the position and/or or ientation of the epsilon-amino group of Lys-80 at the active site to f unction efficiently as a general-base catalyst. Furthermore, the Gly-7 7 and Gly-78 mutant enzymes had markedly decreased thermal stabilities , showing that these two glycyl residues are also critical for the con formational stability of this thermostable enzyme.