The site at which 4-iodoacetamidosalicylate reacts with glutamate dehydrogenases

Abstract

1. Bovine, porcine and chicken liver glutamate dehydrogenases were irreversibly inhibited by a tenfold excess of radioactive 4-iodoacetamidosalicylic acid at pH7.5. 2. Inhibition was accompanied by the covalent incorporation of 1.1 mol of labelled inhibitor/mol of polypeptide chain. Acid hydrolysis yielded N(epsilon)-carboxymethyl-lysine as the sole labelled amino acid. No labelled S-carboxymethylcysteine was recovered from the bovine or porcine enzymes. 3. The labelled bovine enzyme was hydrolysed with trypsin. The radioactivity was found at lysine-126 in a peptide comprising residues 119-130 of the sequence. 4. The amino acid compositions of the tryptic peptides containing labelled lysine from the porcine and chicken enzymes were similar to that of the bovine peptide.

The reaction of a histidine residue in glutamate dehydrogenase with diethyl pyrocarbonate

“…Bovine liver glutamate dehydrogenase was prepared and assayed for activity and protein as previously described (Holbrook et al, 1973). NAD+, NADH, GTP, ADP, ATP and 2-oxoglutarate were obtained from C. F. Boehringer und Soehne G.m.b.H., Mannheim, Germany.…”

The effect of modifying lysine-126 on the physical, catalytic and regulatory properties of bovine liver glutamate dehydrogenase

“…NAD+, NADH, GTP, ADP and 2-oxoglutaric acid were obtained from C. F. Boehringer und Soehne G.m.b.H., Mannheim, Germany. The sources of glutamate dehydrogenase, 4-iodoacetamidosalicylic acid and general laboratory reagents and the methods of enzyme assay and determination ofprotein concentration are all described in the preceding paper (Holbrook et al, 1973).…”

“…15mg of enzyme/ml with 0.8mM-4-iodoacetamidosalicylic acid in 0.067M-NaH2PO4 buffer adjusted to pH7.5 with 5M-NaOH until the required degree of inactivation was obtained. Samples of the incubation mixture were assayed as described in the preceding paper (Holbrook et al, 1973). This method of inhibition resulted in specific alkylation of lysine-126.…”

“…By using 4-iodo[14C]acetamidosalicylate it was found that the reaction was still as specific for lysine at both pH 8.5 and 9.0 as it was at pH7.5. Carboxymethyl-lysine was isolated after acid hydrolysis of the protein by using the method described in the preceding paper (Holbrook et al, 1973). Since 30mM-2-oxoglutarate prevented inhibition at pH 8.5 as it did at pH7.5, it is probable that the lysine alkylated is lysine-126 at all the pH values used.…”

“…The preceding paper (Holbrook et al, 1973) describes the labelling of bovine liver glutamate dehydrogenase [L-glutamate-NAD(P) oxidoreductase (deaminating), EC 1.4.1.3] by 4-iodoacetamidosalicylic acid. Theinhibitoris specificallyincorporated at lysine-126 of the sequence (Moon et al, 1972), causing loss of the enzyme activity.…”