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, 7112-7118]. 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 [3H]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)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TA-5'-DP) modifies Cys-319 in this enzyme [Batra, S. P., & Colman, R. F. (1986) Biochemistry 25, 3508-3515].(ABSTRACT TRUNCATED AT 250 WORDS)
Ozturk
D H
DH
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D000345:Affinity Labels / Q000737:chemistry*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry*; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000032:analysis; D013820:Thermolysin / Q000737:chemistry; D013873:Thionucleotides / Q000737:chemistry*; D014357:Trypsin
D000345:Affinity Labels; D000596:Amino Acids; D013438:Sulfhydryl Compounds; D013873:Thionucleotides; C058333:8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D005969:Glutamate Dehydrogenase; D014357:Trypsin; D013820:Thermolysin; D003545:Cysteine