Identification of lysine 74 in the pyruvate binding site of alanine dehydrogenase from Bacillus subtilis. Chemical modification with 2,4,6-trinitrobenzenesulfonic acid, n-succinimidyl 3-(2-pyridyldithio)propionate, and 5'-(p-(fluorosulfonyl)benzoyl)adenosine.
L-Alanine dehydrogenase from Bacillus subtilis was inactivated with two different lysine-directed chemical reagents, i.e. 2,4, 6-trinitrobenzenesulfonic acid and N-succinimidyl 3-(2-pyridyldithio)propionate. In both cases, the inactivation followed pseudo first-order kinetics, with a 1:1 stoichiometric ratio between the reagent and the enzyme subunits. Partial protection of the active site from inactivation could be obtained by each of the substrates, NADH or pyruvate, but complete protection could only be achieved in the presence of the ternary complex E.NADH. pyruvate. The nucleotide analogue of NADH, 5'-(p-(fluorosulfonyl)benzoyl)adenosine was also used for affinity labeling of the enzyme active site. Differential peptide mapping, performed both in the presence and in the absence of the substrates, followed by reversed phase high performance liquid chromatography separation, diode-array analysis, mass spectrometry, and N-terminal sequencing of the resulting peptides, allowed the identification of lysine 74 in the active site of the enzyme. This residue, which is conserved among all L-alanine dehydrogenases, is most likely the residue previously postulated to be necessary for the binding of pyruvate in the active site. Surprisingly, this residue and the surrounding conserved residues are not found in amino acid dehydrogenases like glutamate, leucine, phenylalanine, or valine dehydrogenases, suggesting that A-stereospecific amino acid dehydrogenases such as L-alanine dehydrogenase could have evolved apart from the B-stereospecific amino acid dehydrogenases.
Delforge
D
D
Laboratory of Cellular Biochemistry, Facultés Universitaires Notre-Dame de la Paix, 61 rue de Bruxelles, B-5000 Namur, Belgium.
Devreese
B
B
Dieu
M
M
Delaive
E
E
Van Beeumen
J
J
Remacle
J
J
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology; D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000494:pharmacology; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism*; D016415:Sequence Alignment; D013054:Spectrophotometry, Atomic; D013388:Succinimides / Q000494:pharmacology; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D013388:Succinimides; D009243:NAD; C018151:N-succinimidyl 3-(2-pyridyldithio)propionate; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D019289:Pyruvic Acid; D014302:Trinitrobenzenesulfonic Acid; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase; D000241:Adenosine