MECHANISMS OF INHIBITION OF AMIDO PHOSPHORIBOSYLTRANSFERASE FROM MOUSE L1210 LEUKEMIA-CELLS

Amido phosphoribosyltransferase (amido PRTase) catalyses the first ste p of the pathway for de novo biosynthesis of purine nucleotides. The e nzyme is subject to inhibition by purine nucleoside 5'-monophosphates (AMP, IMP, and GMP), by dihydrofolate polyglutamates, and by the antif olate piritrexim [Sant, M. E., Lyons, S. D., Phillips, L., & Christoph erson, 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 mu M for 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) an d 421 +/- 193 mu M for L-glutamine with P-Rib-PP bound first with posi tive cooperativity for interaction with a second site on the catalytic ally active dimer (interaction factor of 0.247 +/- 0.042). Analysis of inhibition patterns for amido PRTase shows that the antifolate piritr exim is a noncompetitive inhibitor bound with positive cooperativity a t two allosteric sites of an inactive dimer with a dissociation consta nt of 66.0 +/- 17.8 mu M for interaction with the free enzyme and an i nteraction factor of 0.187 +/- 0.113 with P-Rib-PP as the varied subst rate. With L-glutamine as the varied substrate, a dissociation constan t of 62.3 +/- 15.6 mu M for interaction with the enzyme-P-Rib-PP compl ex and an interaction factor of 0.0958 +/- 0.0585 mu M were obtained. AMP binds as a competitive inhibitor with respect to P-Rib-PP with a d issociation constant of 40.0 +/- 8.1 mu M for interaction with the fre e 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 o f partially purified amido PRTase showed three molecular forms of the enzyme: an inactive tetramer (10.2 S) formed in the presence of AMP, a n 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 cont aining high levels of dihydrofolate polyglutamates.