STABLE CARBOCYCLIC ANALOG OF 5-PHOSPHORIBOSYL-1-PYROPHOSPHATE TO PROBE THE MECHANISM OF CATALYSIS AND REGULATION OF GLUTAMINE PHOSPHORIBOSYLPYROPHOSPHATE AMIDOTRANSFERASE

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase catalysi s and regulation were studied using a new stable carbocyclic analog of PRPP, dihydroxy-4-beta-cyclopentane-methanol-5-phosphate (cPRPP). Alt hough cPRPP competes with PRPP for binding to the catalytic C site of the Escherichia coli enzyme, two lines of evidence demonstrate that cP RPP, unlike PRPP, does not promote an active enzyme conformation. Firs t, cPRPP was not able to ''activate'' Cys(1) for reaction with glutami ne or a glutamine affinity analog. The ring oxygen of PRPP may thus be necessary for the conformation change that activates Cys(1) for catal ysis. Second, binding of cPRPP to the C site blocks binding of AMP and GMP, nucleotide end product inhibitors, to this site. However, the bi nding of nucleotide to the allosteric site was essentially unaffected by cPRPP in the C site. Since it is expected that nucleotide inhibitor s would bind with low affinity to the active enzyme conformation, the nucleotide binding data support the conclusion that cPRPP does not act ivate the enzyme.