Regulation and Mechanism of Phosphoribosylpyrophosphate Synthetase: V. INHIBITION BY END PRODUCTS AND REGULATION BY ADENOSINE DIPHOSPHATE

Abstract

Abstract Phosphoribosylpyrophosphate (PRPP) synthetase participates in the biosynthesis in bacteria of purine nucleotides, pyrimidine nucleotides, tryptophan, histidine, and the pyridine nucleotide coenzymes. A kinetic study of the interaction of highly purified PRPP synthetase from Salmonella typhimurium with end products and other potential metabolite effectors has been conducted. There is no evidence for cooperative homotropic substrate interactions in the presence or absence of inhibitors. ADP, which is a much more effective inhibitor as a function of concentration than any other nucleotide, displays a complex inhibition pattern. A detailed steady state kinetic analysis of ADP inhibition is consistent with competition at the ATP site and formation of a dead-end E(ADP) (PRPP) complex, when subsaturating concentrations of ribose-5-P are used or when the reverse reaction is studied. However, ADP also alters PRPP synthetase in such a fashion as to bring about pronounced substrate inhibition by ribose-5-P. This inhibition is not purely the result of formation of a E(ADP) (ribose-5-P) complex, but appears to be the consequence of binding of ADP and possibly ribose 5-P at sites that are distinct from the substrate sites. Other nucleotide inhibitors do not bring about substrate inhibition. Low concentrations of Ca2+ ions bring about substrate inhibition in the absence of ADP and partially desensitize PRPP synthetase to the effect of ADP. Purine nucleotides are much more effective inhibitors than pyrimidine nucleotides; both are purely competitive with ATP. Kinetic analysis of inhibition by pairs of nucleotide inhibitors (except ADP) indicates that, although these inhibitors are competitive with ATP, they exert synergistic effects on the action of each other and do not bind in a mutually exclusive fashion. Histidine and tryptophan do not inhibit purified PRPP synthetase under any conditions examined. Deoxynucleotides, pyridine nucleotides, cyclic adenosine 3',5'-monophosphate, 2'- or 3'-pyrophosphoryl guanosine 5'-diphosphate, and intermediates of glycolysis and the pentose cycle are either very weak inhibitors or without effect on PRPP synthetase activity. Although the physiological significance of the various kinetic effects cannot be established by these studies alone, the findings are consistent with the proposals that (a) PRPP synthetase is subject to regulation by the ATP/ADP ratio and hence the supply of metabolic energy, (b) the sensitivity to ATP/ADP ratio is modified by the concentrations of ribose-5-P in the cell, and (c) the enzyme may be controlled by the total pool of nucleotide end products, particularly purine nucleotides. The observation that PRPP synthetase is subject to specific repressive control by pyrimidines (White, M. N., Olszowy, J., and Switzer, R. L. (1971) J. Bacteriol. 108, 122) suggests that PRPP synthesis may be controlled in bacteria by regulatory complementation between feedback inhibition by purine nucleotides and repression by pyrimidine nucleotides.