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 catalytically important lysyl residue, is highly conserved in NAD(P)+-dependent amino acid dehydrogenases. To elucidate functional roles of the glycyl residues in this conserved sequence Gly-77, Gly-78, and Gly-79 of the recombinant leucine dehydrogenase from Bacillus stearothermophilus have been individually replaced with Ala by site-directed mutagenesis. All of the mutant enzymes had Michaelis constants for alpha-keto-iso-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 inactivation by pyridoxal 5'-phosphate and the microenvironment of aromatic residues, 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 glycyl residues are important for fine-tuning of the position and/or orientation of the epsilon-amino group of Lys-80 at the active site to function efficiently as a general-base catalyst. Furthermore, the Gly-77 and Gly-78 mutant enzymes had markedly decreased thermal stabilities, showing that these two glycyl residues are also critical for the conformational stability of this thermostable enzyme.
Sekimoto
T
T
Institute of Scientific and Industrial Research, Osaka University.
Fukui
T
T
Tanizawa
K
K
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002208:Caproates / Q000494:pharmacology; D002384:Catalysis; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D005998:Glycine / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D050803:Leucine Dehydrogenase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate; D013050:Spectrometry, Fluorescence
D002208:Caproates; C001916:alpha-ketocaproic acid; D011732:Pyridoxal Phosphate; D000594:Amino Acid Oxidoreductases; D050803:Leucine Dehydrogenase; D005998:Glycine