Identification of histidyl peptide labeled by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate in an ADP regulatory site of glutamate dehydrogenase.
Bovine liver glutamate dehydrogenase reacts covalently with 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of 1 mol reagent/mol enzyme subunit and loss of one of the two ADP sites of native enzyme [S. P. Batra and R. F. Colman, J. Biol. Chem. 261, 15565-15571 (1986)]. Incorporation of reagent is prevented specifically by ADP. The modified enzyme has now been digested with trypsin. The nucleotidyl peptide has been purified by chromatography on phenylboronate-agarose, followed by reverse-phase HPLC. On the basis of amino acid composition following acid hydrolysis, and gas-phase sequencing, the modified tryptic peptide was established as Ala-Gln-His-Ser-Gln-His-Arg, corresponding to amino acids 80-86 of the known glutamate dehydrogenase primary structure. The evidence presented indicates that the target amino acid attacked by 2-BDB-TAMP is histidine-82 and that this residue is located within the high-affinity ADP-activating site of glutamate dehydrogenase. In the course of this work, it was found that the positions of Gln84 and His85 had been reported as reversed in the revised sequence of bovine liver glutamate dehydrogenase [J. H. Julliard and E. L. Smith, J. Biol. Chem. 254, 3427-3438 (1979)]. Three additional corrections are here reported in the amino acid sequence of the native enzyme on the basis of gas-phase sequencing of other peptides purified by HPLC: Asp168 (not Asn); His221-Gly222 (not Gly-His); and Glu355 (not Gln).
Batra
S P
SP
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Lark
R H
RH
Colman
R F
RF
D000244:Adenosine Diphosphate / Q000032:analysis*; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000032:analysis; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006639:Histidine / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification*; D013873:Thionucleotides; D014357:Trypsin
D000345:Affinity Labels; D000596:Amino Acids; D010446:Peptide Fragments; D013873:Thionucleotides; D000249:Adenosine Monophosphate; D006639:Histidine; D000244:Adenosine Diphosphate; C048059:2-((4-bromo-2,3-dioxobutyl)thio)adenosine 5'-monophosphate; D005969:Glutamate Dehydrogenase; D014357:Trypsin