The effects of an acetate-sensitive anion binding site on NADPH binding in glutamate dehydrogenase.
The nature of a general anion binding site that regulates NADPH binding to L-glutamate dehydrogenase has been explored. Dissociation constants for the enzyme-NADPH complex were measured by difference spectroscopy in the presence of phosphate, pyrophosphate, ADP and acetate ions. Whereas two molecules of phosphate, binding in a cooperative fashion, raise the Kd of the enzyme-NADPH complex 50-fold from 2.3 microM, a single pyrophosphate raises the Kd only 23-fold, disproving the notion that the anion binding site is simply the pyrophosphate binding site of NADPH. ADP raises the Kd of the enzyme-NADPH complex 2-fold for a given phosphate concentration, and formation of the enzyme-ADP complex is itself interfered with by phosphate and pyrophosphate, indicating that these anions interact with the same anion binding site. Acetate ion acts in a manner opposite to that of phosphate, pyrophosphate and ADP and reverses the weakening effect that these ions exert on NADPH binding, returning the Kd of the enzyme-NADPH complex to 2.3 microM. In the absence of these anions, however, acetate exerts no measurable effect on the Kd, suggesting an allosteric mechanism.
D000085:Acetates / Q000494:pharmacology*; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000495:Allosteric Site; D000838:Anions / Q000378:metabolism*; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D009249:NADP / Q000378:metabolism*; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects
D000085:Acetates; D000838:Anions; D010710:Phosphates; D009249:NADP; D000244:Adenosine Diphosphate; D005969:Glutamate Dehydrogenase