Identification of Lysine 74 in the Pyruvate Binding Site of Alanine Dehydrogenase from Bacillus subtilis

Abstract

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.

Mycobacterium smegmatisl-Alanine Dehydrogenase (Ald) Is Required for Proficient Utilization of Alanine as a Sole Nitrogen Source and Sustained Anaerobic Growth

“…It also exhibits approximately 60% identity to isozymes of Streptomyces coelicolor (33) and B. subtilis (37). The multiple alignment revealed two highly conserved regions with the consensus sequences K 73 VKEP 77 and v 173 iGgGtaGyNAAriA-G mGa-VTvlDvn 200 , which correspond to the enzyme active site for pyruvate binding (13) and the NAD cofactor binding region (2), respectively. Ald activity is regulated by nutrient supplementation, oxygen availability, and growth stage.…”

Purification and characterization of oxalate oxidase from wheat seedlings

“…Modification of lysines 2,4,6-Trinitrobenzene sulfonic acid (TNBS) is a modifier of lysine residues (Delforge et al 1997;Kotsira and Clonis 1998;Liu et al 1999). In the presence of TNBS, OxO activity was inhibited in the first 2 min, and then declined slowly (Fig.…”

Probing protein structure by amino acid‐specific covalent labeling and mass spectrometry

“…Remacle and co-workers investigated the amino acid residues that might be involved in the substrate binding and catalysis of L-Alanine dehydrogenase from Bacillus subtilis with chemical modification and kinetic studies (Delforge et al, 1997). Amino groups were modified by reactions with 2,4,6-trinitrobenzenesulfonic acid (TNBS), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 5′-(p-(fluorosulfonyl)benzoyl) adenosine (FSBA).…”