Activation of bovine liver glutamate dehydrogenase by covalent reaction of adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] with arginine-459 at an ADP regulatory site.
Bovine liver glutamate dehydrogenase is an allosteric enzyme which is activated by ADP. The affinity label adenosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thiophosphate] (AMPSBDB), a new ADP analog featuring a reactive group at a position equivalent to that of the pyrophosphate, reacts with this glutamate dehydrogenase to yield enzyme containing about 0.9 mol/mol of enzyme subunit. The reaction results in a time-dependent irreversible activation of the enzyme. Glutamate dehydrogenase (8.9 microM subunit) modified with 10-60 microM AMPSBDB is about 3.2-fold more active than native enzyme. The modified enzyme is still inhibited by GTP and by high concentrations of NADH, but is no longer activated by ADP. The addition to the reaction mixture of (a) NADH or alpha-ketoglutarate; (b) GTP + NADH; or (c) alpha-ketoglutarate + NADH has little effect on the functional changes produced by AMPSBDB; whereas, the reaction is prevented by ADP. Purification of labeled peptide from proteolytic and chemical digests of [2-3H]AMPSBDB-modified enzyme leads to identification of Arg459 as the target amino acid. We conclude that AMPSBDB functions as an ADP mimic covalently bound to Arg459 within the ADP activator site of the allosteric bovine liver glutamate dehydrogenase.
Wrzeszczynski
K O
KO
Department of Chemistry and Biochemistry, University of Delaware, Newark 19716.
Colman
R F
RF
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D000345:Affinity Labels / Q000737:chemistry / Q000494:pharmacology*; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D017421:Sequence Analysis; D013873:Thionucleotides / Q000737:chemistry / Q000494:pharmacology*
D000345:Affinity Labels; D008024:Ligands; D010446:Peptide Fragments; D013873:Thionucleotides; C086304:adenosine 5'-O-(S-(4-bromo-2,3-dioxobutyl))thiophosphate; D009243:NAD; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D005969:Glutamate Dehydrogenase