Affinity labeling of the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase by 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate.
Bovine liver glutamate dehydrogenase reacts covalently with the new adenosine analogue 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate with incorporation of about 1 mol of reagent/mol of enzyme subunit. Modified enzyme completely loses its normal ability to be inhibited by high concentrations of reduced diphosphopyridine nucleotide (DPNH) (greater than 100 microM), which binds at a regulatory site distinct from the catalytic site; however, the modified enzyme retains its full activity when assayed at 100 microM DPNH in the absence of allosteric compounds. The enzyme is still activated by ADP, is inhibited by GTP (albeit at higher concentrations), and binds 1.5-2 mol of [14C]GTP/subunit. A plot of initial velocity vs. DPNH concentration for the modified enzyme, in contrast to the native enzyme, followed Michaelis-Menten kinetics. The rate constant (k) for loss of DPNH inhibition (as measured at 0.6 mM DPNH) exhibits a nonlinear dependence on reagent concentration, suggesting a reversible binding of reagent (Kd = 0.19 mM) prior to irreversible modification. At 0.1 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate, k = 0.036 min-1 and is not affected by alpha-ketoglutarate, 100 microM DPNH, or GTP alone but is decreased to 0.0094 min-1 by 5 mM DPNH and essentially to zero by 5 mM DPNH plus 100 microM GTP. Incorporation after incubation with 0.25 mM 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate for 2 h at pH 7.1 is 1.14 mol/mol of subunit in the absence but only 0.24 mol/mol of subunit in the presence of DPNH plus GTP.(ABSTRACT TRUNCATED AT 250 WORDS)
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D001665:Binding Sites; D005969:Glutamate Dehydrogenase / Q000037:antagonists & inhibitors*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D046911:Macromolecular Substances; D009243:NAD / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013873:Thionucleotides
D000345:Affinity Labels; D046911:Macromolecular Substances; D009711:Nucleotides; D013873:Thionucleotides; D009243:NAD; D000244:Adenosine Diphosphate; C042298:6-((4-bromo-2,3-dioxobutyl)thio)-6-deaminoadenosine 5'-diphosphate; D005969:Glutamate Dehydrogenase