Distance relationships between the catalytic site labeled with 4-(iodoacetamido)salicylic acid and regulatory sites of glutamate dehydrogenase.
The distance between the catalytic site on bovine liver glutamate dehydrogenase labeled with 4-(iodoacetamido)salicylic acid (ISA) and the adenosine 5'-diphosphate (ADP) activatory site occupied by the analogue 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-diphosphate (TNP-ADP) was evaluated by energy transfer. Native enzyme and enzyme containing about 1 mol of acetamidosalicylate/mol of subunit bind about 0.5 mol of TNP-ADP/mol of subunit, and TNP-ADP competes for binding with ADP to native and modified enzyme, indicating that the analogue is a satisfactory probe of the ADP site. From the quenching of acetamidosalicylate donor fluorescence upon addition of TNP-ADP, an average distance of 33 A was determined between the catalytic and ADP sites. The fluorescent nucleotide analogue 5'-[p-(fluorosulfonyl)benzoyl]-2-aza-1,N6-ethenoadenosine (5'-FSBa epsilon A) reacts covalently with glutamate dehydrogenase to about 1 mol/peptide chain. As compared to native enzyme, the SBa epsilon A-enzyme exhibits decreased sensitivity to GTP inhibition but retains its catalytic activity as well as its ability to be activated by ADP and inhibited by high concentrations of NADH. Complete protection against decreased sensitivity to GTP inhibition is provided by GTP in the presence of NADH. It is concluded that 5'-FSBa epsilon A modifies a GTP site on glutamate dehydrogenase. The distance of 23 A between the catalytic site labeled with ISA and a GTP site labeled with 5'-FSBa epsilon A was measured from the quenching of salicylate donor fluorescence in the presence of the SBa epsilon A acceptor on a doubly labeled enzyme. The average distance between the ADP and GTP sites was previously measured as 18 A [Jacobson, M. A., & Colman, R. F. (1983) Biochemistry 22, 4247-4257], indicating that the regulatory sites of glutamate dehydrogenase are closer to each other than to the catalytic site.
D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000495:Allosteric Site; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004735:Energy Transfer; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007460:Iodoacetamide / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D012459:Salicylates / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
C024128:5'-(4-fluorosulfonylbenzoyl)-2-aza-1,N(6)-ethenoadenosine; D000596:Amino Acids; D005456:Fluorescent Dyes; D012459:Salicylates; C005487:4-iodoacetamidosalicylic acid; D000244:Adenosine Diphosphate; C036745:2',3'-(O-(2,4,6-trinitrocyclohexadienylidine))adenosine 5'-diphosphate; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase; D000241:Adenosine; D007460:Iodoacetamide