Investigation of the actives site of alanine dehydrogenase from aeromonas hydrophila by chemical modification

Abstract

Alanine dehydrogenase from Aeromonas hydrophila was chemically modified with a series of group -specific reagents to identify essential amino acid residues. Incubation of the enzyme with to 10 mM of chloramine T (CT), diethylpyrocarbonate (DEPC), phenylglyoxal (PG), and 2,4,6-trinitrobenzenesulfonic acid (TNBS) which were specific for methionine, histidine, arginine, and lysine, respectively, led to complete loss of enzyme activity. In addition N-acetylimidazole (NAI), which is specific for tyrosine, reduced the enzyme activity to 88.9%, while dithiothreitol (DTT), a modifier of cysteine, did not affect the enzyme activity. About 30-35 % of the enzyme activity could be regained when pyruvate or NADH protection against the modification of DEPC, PG and TNBS were performed. Moreover, up to 90% of the residual activity was found when the enzyme was protected by both pyruvate and NADH. No significant protection was observed when CT was used as the modifying reagent. The result suggested that histidine, arginine, and lysine should directly involve in the active site of alanine dehydrogenase while methionine should not. The pattern of inactivation by DEPC and PG proceeded through pseudo-first-order kinetics. The simple bimolecular mechanism was observed with a 1:1 stoichiometric ratio of mole reagent per mole enzyme subunit. Substrate protection pattern indicated that DEPC -modified histidine and PG -modified arginine should be parts of the active site. Since kinetic mechanism of this enzyme is sequential order binary-ternary, which pyruvate can bind to the binding site after NADH is already fixed to the active site, modified histidine and arginine were proposed to locate in the pyruvate bin d in g site. Peptide mapping performed in both native and DEPC-modified enzymes by reverse phase high performance liquid chromatography and N- terminal amino acid sequencing suggested that H is-9 5 might be an essential amino acid residue in pyruvate binding site.