Identification of cysteine-319 as the target amino acid of 8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5 prime -triphosphate in bovine liver glutamate dehydrogenase

Journal Article · Tue Jul 23 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00243a013· OSTI ID:5032203

Ozturk, D H; Colman, R F ^[1]

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1. Univ. of Delaware, Newark (United States)

The affinity label 8-((4-bromo-2,3-dioxobutyl)thio)adenosine 5{prime}-triphosphate (8-BDB-TA-5{prime}-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. The modified enzyme was shown to contain only 5 free sulfhydryl groups upon 5,5{prime}-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 ({sup 3}H)acetic acid. In contrast, only 5(carboxymethyl)cysteinyl peptides were detected in 8-BDB-TA-5{prime}-TP-modified enzyme. When carboxymethylated modified and unmodified enzymes were digested with thermolysin, 6 peptide sequences containing (carboxymethyl)cysteine which was absent in the modified enzyme was determined to be Cys-319, leading to the conclusion that 8-BDB-TA-5{prime}-TP reacts with Cys-319, thereby preventing it from subsequent reaction with radioactive iodoacetate.

• OSTI ID: 5032203
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:29; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETIC ACID
AMINE OXIDASES
AMINO ACID SEQUENCE
AMINO ACIDS
ATP
CARBOXYLIC ACIDS
CHROMATOGRAPHY
CYSTEINE
ENZYMES
GLUTAMIC ACID
HYDROGEN COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
MOLECULAR STRUCTURE
MONOCARBOXYLIC ACIDS
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
OXIDOREDUCTASES
RADIOCHROMATOGRAPHY
SEPARATION PROCESSES
THIOLS
TRITIUM COMPOUNDS