Mechanisms of Inhibition of Amido Phosphoribosyltransferase from Mouse L1210 Leukemia Cells

Amido phosphoribosyltransferase (amido PRTase) catalyses the first step of the pathway for de novo biosynthesis of purine nucleotides. The enzyme is subject to inhibition by purine nucleoside 5‘-monophosphates (AMP, IMP, and GMP), by dihydrofolate polyglutamates, and by the antifolate piritrexim [Sant, M. E., Lyons, S. D., Phillips, L., & Christopherson, R. I. (1992) J. Biol. Chem. 267, 11038−11045). Using a coupled radioassay, we have determined the substrate dissociation constants as 80.4 ± 13.2 μM for 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) and 421 ± 193 μM for l-glutamine with P-Rib-PP bound first with positive cooperativity for interaction with a second site on the catalytically active dimer (interaction factor of 0.247 ± 0.042). Analysis of inhibition patterns for amido PRTase shows that the antifolate piritrexim is a noncompetitive inhibitor bound with positive cooperativity at two allosteric sites of an inactive dimer with a dissociation constant of 66.0 ± 17.8 μM for interaction with the free enzyme and an interaction factor of 0.187 ± 0.113 with P-Rib-PP as the varied substrate. With l-glutamine as the varied substrate, a dissociation constant of 62.3 ± 15.6 μM for interaction with the enzyme−P-Rib-PP complex and an interaction factor of 0.0958 ± 0.0585 μM were obtained. AMP binds as a competitive inhibitor with respect to P-Rib-PP with a dissociation constant of 40.0 ± 8.1 μM for interaction with the free enzyme and as a noncompetitive inhibitor with respect to l-glutamine with a dissociation constant of 16.4 ± 5.2 mM for interaction with the enzyme−P-Rib-PP complex. Sucrose density gradient centrifugation of partially purified amido PRTase showed three molecular forms of the enzyme: an inactive tetramer (10.2 S) formed in the presence of AMP, an active dimer (6.7 S) formed with P-Rib-PP, and an inactive dimer (7.2 S) with piritrexim. The latter species may predominate in cells containing high levels of dihydrofolate polyglutamates.