Isolation and identification of cysteinyl peptide labeled by 6- [( 4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate in the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase.
6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate (6-BDB-TADP) has been shown to react at the reduced diphosphopyridine nucleotide (DPNH) inhibitory site of bovine liver glutamate dehydrogenase with incorporation of 1 mol of reagent/mol of enzyme subunit [Batra, S. P., & Colman, R. F. (1984) Biochemistry 23, 4940-4946]. The modified enzyme had lost one of the six free sulfhydryl groups per enzyme subunit as detected by 5,5'-dithiobis(2-nitrobenzoate). In the unmodified enzyme digested with trypsin, six cysteinyl peptides labeled with [14C]iodoacetic acid were detected by high-performance liquid chromatography (HPLC), whereas only five were observed in the 6-BDB-TADP-modified enzyme. A cysteinyl peptide has been isolated from modified enzyme digested with trypsin and chymotrypsin. Purification of the nucleotidyl peptide was accomplished by chromatography on phenyl boronate-agarose, followed by gel filtration on Sephadex G-25 and Bio-Gel P-4 in 50 mM ammonium bicarbonate, pH 8.0. The modified peptides were finally purified by HPLC on a C18 column using 0.1% trifluoroacetic acid with an acetonitrile gradient. By comparison of the amino acid composition and N-terminal residue of the isolated peptide with the known amino acid sequence of the enzyme, the peptide in the DPNH inhibitory site labeled by 6-BDB-TADP has been identified as the 19-membered fragment from Glu-311 to Lys-329. A unique residue, Cys-319, was identified as the reactive amino acid within the DPNH inhibitory site.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D009243:NAD / Q000494:pharmacology*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification*; D013873:Thionucleotides; D014357:Trypsin
D000345:Affinity Labels; D000596:Amino Acids; D002250:Carbon Radioisotopes; D010446:Peptide Fragments; D013873:Thionucleotides; D009243:NAD; D000244:Adenosine Diphosphate; C042298:6-((4-bromo-2,3-dioxobutyl)thio)-6-deaminoadenosine 5'-diphosphate; D005969:Glutamate Dehydrogenase; D014357:Trypsin; D003545:Cysteine