NADPH and substrates protect ATP-citrate lyase from thermal and proteolytic inactivation.

Three distinct classes of compounds can effectively protect ATP-citrate lyase from thermal and proteolytic inactivation. For example, the substrate citrate is the prototype of one of these classes, in this case protecting the enzyme by binding apparently to the active site and causing a conformational change. Other compounds with suitably spaced negative charges (e.g. fructose 1,6bisphosphate, phosphoenolpyruvate) also protect, presumably by interaction with the same site. Coenzyme A, another substrate, also affords protection by binding at the active site. By testing the effects of different fragments of the coenzyme A molecule we have shown that the major portion of this coenzyme that is recognized is the adenosine moiety. In particular, the 3’phosphoryl group on the ribose residue of coenzyme A is an absolute requirement for binding to this site. Further to these effects of substrates, we made the unexpected discovery that nucleotides with a 2’-phosphoryl group also afforded specific protection by interaction with a separate binding site. By far the most effective. 2’-nucleotide is NADPH, which at physiological concentrations gives marked protection of ATPcitrate lyase from degradation. Significantly, NADP+ is much less effective and neither NAD’ nor NADH afford any protection. Furthermore, the absence of effect of nucleotides with 2‘,3’-cyclic phosphates clearly distinguishes between the coenzyme A and NADPH binding sites and points to a high degree of specificity for each. Although all three classes of compounds interact with different sites, they appear to act by promoting an equivalent conformational change in the protein. This structural transition allows the enzyme to be proteolytically nicked, but full enzymatic activity is retained and further proteolysis is blocked. The observed effects of substrates and NADPH may play a supportive role in limiting degradation rates in vivo, since the level of this enzyme is under hormonal control.