IDENTIFICATION OF CYSTEINE-319 AS THE TARGET AMINO-ACID OF 8-[(4-BROMO-2,3-DIOXOBUTYL)THIO]ADENOSINE 5'-TRIPHOSPHATE IN BOVINE LIVER GLUTAMATE-DEHYDROGENASE

The affinity label 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 5'-triphosphate (8-BDB-TA-5'-TP) has been shown to react with bovine liver glutamate dehydrogenase in the region of the GTP-dependent NADH inhibitory site with incorporation of about 1 mol of reagent/mol of subunit [Ozturk, D. H., Safer, D., & Colman, R. F. (1990) Biochemistry 29, 7112-71181. The modified enzyme was shown to contain only 5 free sulfhydryl groups upon 5,5'-dithiobis(2-nitrobenzoate) titration as compared with 6 in the unmodified enzyme. In the unmodified enzyme digested with trypsin, 6 cysteinyl peptides were detected by high-performance liquid chromatography upon treatment with iodo[H-3]acetic acid. In contrast, only 5 (carboxymethyl)cysteinyl peptides were detected in 8-BDB-TA-5'-TP-modified enzyme. When carboxymethylated modified and unmodified enzymes were digested with thermolysin, 6 peptide sequences containing (carboxymethyl)cysteine were obtained in the unmodified enzyme, but only 5 were observed in the modified enzyme. The (carboxymethyl)cysteine which was absent in the modified enzyme was determined to be Cys-319, leading to the conclusion that 8-BDB-TA-5'-TP reacts with Cys-319, thereby preventing it from subsequent reaction with radioactive iodoacetate. It was previously reported that 6-[(4-bromo-2,3-dioxobutyl) thiol-6-deaminoadenosine 5'-diphosphate (6-BDB-TA-5'-DP) modifies Cys-319 in this enzyme [Batra, S. P., & Colman, R. F. (1986) Biochemistry 25, 3508-35151. Evidence is here presented that the reaction product is different for the two nucleotide analogues: while Cys-319 reacts at the methylene group of 6-BDB-TA-5'-DP by displacement of bromide, the same cysteine appears to attack the carbonyl group of 8-BDB-TA-5'-TP, leading to a thiohemiacetal adduct.