AFFINITY LABELING OF AN ALLOSTERIC GTP SITE OF BOVINE LIVER GLUTAMATE-DEHYDROGENASE BY 5'-PARA-FLUOROSULFONYLBENZOYLGUANOSINE

In the presence of DPNH, native glutamate dehydrogenase binds, with markedly different affinities, 2 mol of the allosteric inhibitor GTP per peptide chain. In contrast, only 1 mol of GTP is bound in the absence of coenzyme. Incubation of enzyme with the guanosine nucleotide analogue 5'-p-fluorosulfonylbenzoylguanosine does not affect the intrinsic catalytic activity of the enzyme as measured in the absence of regulatory compounds, but leads to a progressive decrease in the sensitivity to inhibition by GTP. The modified enzyme binds only 1 mol of GTP per peptide chain, in the presence or absence of DPNH, implying that reaction with 5'p fluorosulfonylbenzoylguanosine eliminates one of the allosteric sites for GTP. In contrast, the Michaelis constants for substrates and the ability of the enzyme to be inhibited by high concentrations of DPNH are not appreciably changed by the modification reaction. Although the affinity for the activator ADP is not altered, the maximum extent of activation is decreased. The rate constant for reaction of glutamate dehydrogenase with 5'-p-fluorosulfonylbenzoylguanosine has been measured from the time dependence of the decreased inhibition by 1.1 μM GTP; this rate constant is specifically and strikingly decreased by low concentrations of GTP in the presence of reduced coenzyme but not by substrates, DPNH alone or ADP either with or without DPNH. The extent of covalent incorporation of radioactive 5'-sulfonylbenzoylguanosine is directly proportional to the percent decrease in GTP inhibition, a maximum alteration in sensitivity to GTP being observed when approximately 2 mol of 5'-sulfonylbenzoylguanosine is incorporated per enzyme subunit. Only 1 mol of reagent per peptide chain is covalently bound in the presence of GTP and reduced coenzyme, which protect the enzyme against the decreased response to GTP but do not prevent the decreased activation by ADP. In contrast, about 2 mol of reagent per enzyme subunit is incorporated in the presence of ADP and DPNH, which protect neither against the reduced response to ADP nor to GTP. These results suggest that incorporation of 1 mol of 5'-p-sulfonylbenzoylguanosine specifically causes elimination of one of the GTP sites of the native enzyme and that this change is responsible for the decreased sensitivity to GTP inhibition. Incorporation of the second mole of 5'-p-sulfonylbenzoylguanosine may occur at a site distinct from the recognized allosteric sites and indirectly cause a change in the extent of activation by ADP. © 1979, American Chemical Society. All rights reserved.