title: Mechanisms of inhibition of amido phosphoribosyltransferase from mouse L1210 leukemia cells.
abstract: 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 microM for 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP) and 421 +/- 193 microM 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 microM 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 microM for interaction with the enzyme-P-Rib-PP complex and an interaction factor of 0.0958 +/- 0.0585 microM were obtained. AMP binds as a competitive inhibitor with respect to P-Rib-PP with a dissociation constant of 40.0 +/- 8.1 microM 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.
authors.affiliation: Department of Biochemistry, University of Sydney, New South Wales, Australia.
authors.forename: S L
authors.initials: SL
authors.lastname: Schoettle
authors.forename: L B
authors.initials: LB
authors.lastname: Crisp
authors.forename: E
authors.initials: E
authors.lastname: Szabados
authors.forename: R I
authors.initials: RI
authors.lastname: Christopherson
mesh_terms: D000249:Adenosine Monophosphate / Q000494:pharmacology*; D000582:Amidophosphoribosyltransferase / Q000037:antagonists & inhibitors*; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D001667:Binding, Competitive; D002499:Centrifugation, Density Gradient; D002855:Chromatography, Thin Layer; D007700:Kinetics; D007939:Leukemia L1210 / Q000201:enzymology*; D051379:Mice; D011743:Pyrimidines / Q000494:pharmacology*; D011869:Radioligand Assay; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D000970:Antineoplastic Agents; D011743:Pyrimidines; D000249:Adenosine Monophosphate; D000582:Amidophosphoribosyltransferase; C024530:piritrexim