Affinity labeling of a regulatory site of bovine liver glutamate dehydrogenase

Article abstract of DOI:10.1021/bi00675a010

A new adenosine analog 3' p fluorosulfonyl benzoyladenosine (3' FSBA), has been synthesized which reacts covalently with bovine liver glutamate dehydrogenase. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of NADH. Both of these effects are irreversibly decreased upon incubation of the enzyme with the adenosine analog, 3' FSBA, while the intrinsic enzymatic activity as measured in the absence of regulatory compounds remains unaltered. A plot of the rate constant for modification as a function of the 3' FSBA concentration is not linear, suggesting that the adenosine derivative binds to the enzyme (K(I)=1.0x10^-4M) prior to the irreversible modification. Protection against modification by 3' FSBA is provided by ADP and by high concentrations of NADH, but not by the inhibitor GTP, the substrate α keto glutarate, the coenzyme TPNH, or low concentrations of the coenzyme DPNH. The isolated altered enzyme contains approximately 1 mol of sulfonylbenzoyladenosine per peptide chain, indicating that a single specific regulatory site has reacted with 3' FSBA. The modified enzyme exhibits normal Michaelis constants for its substrates and is still inhibited by GTP, albeit at a higher concentration, but it is not inhibited by high concentrations of NADH. Although ADP does not appreciably activate the modified enzyme, it does (as in the case of the native enzyme) overcome the inhibition of the modified enzyme by GTP. These results suggest that ADP can bind to the modified enzyme, but that its ability to activate is affected indirectly by the modification of the adjacent NADH inhibitory site. It is proposed that the regulatory sites for ADP and NADH are partially overlapping and that 3' FSBA functions as a specific affinity label for the NADH inhibitory site of glutamate dehydrogenase. It is anticipated that 3' p fluorosulfonyl benzoyladenosine may act as an affinity label of other dehydrogenases as well as of other classes of enzymes which use adenine nucleotides as substrates or regulators.