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.

Inhibitors of de Novo Nucleotide Biosynthesis as Drugs

“…Amido Phosphoribosyltransferase (APRTase, P-Rib-PP → PRA). APRTase catalyzes the first committed step of the de novo purine pathway (reaction 1, Figure ) and is inhibited by AMP, IMP, and GMP . The pentaglutamyl derivative of dihydrofolate and the nonclassical antifolate, piritrexim (Figure ), are potent inhibitors of APRTase in vitro .…”

“…APRTase catalyzes the first committed step of the de novo purine pathway (reaction 1, Figure ) and is inhibited by AMP, IMP, and GMP . The pentaglutamyl derivative of dihydrofolate and the nonclassical antifolate, piritrexim (Figure ), are potent inhibitors of APRTase in vitro . De novo purine biosynthesis in mouse L1210 leukemia cells exposed to 1 μM methotrexate is blocked at reactions 1, 3, and 9, presumably due to inhibition of these enzymes by accumulated dihydrofolate polyglutamates .…”

In silico analysis of glycinamide ribonucleotide transformylase inhibition by PY873, PY899 and DIA

“…2,4-Diamino analogs of folic acid have been important in cancer chemotherapy. Despite its complexity, the underlying basis of cell growth inhibition by these compounds relies on their ability to block de novo synthesis of the purine nucleotides, i.e., precursors of DNA (Schoettle et al, 1997). This work is an extension of our previous study where we reported inhibition of amido phosphoribosyltransferase using 2,4-Diamino analogs of folic acid (Batool et al, 2013).…”

Amidophosphoribosyltransferase

“…10 mM, complete inhibition, sigmoidal inhibition curve, competitive vs. 5-phospho-a-d-ribose 1-diphosphate, GMP and AMP together have a synergistic effect on inhibition [3]; <3> 1 mM, approx. 40% inhibition [6]; <9> no inhibition in the presence of 5-phospho-a-d-ribose 1-diphosphate at high concentrations [7]; <6> 1.8 mM, 50% inhibition [8]; <6> 5 mM, 76% inhibition, noncompetive vs. glutamine [13]; <6> 5 mM, 79% and 24% inhibition of aminotransferase and amidotransferase activity at 1 mM 5-phospho-a-d-ribose 1-diphosphate [14]; <11> competitive vs. 5-phospho-a-d-ribose 1-diphosphate [16]; <10> 2.7 mM and 1.2 mM, 50% inhibition of stable and unstable enzyme preparation respectively [17]; <4> 5 mM, 50% inhibition [18]; <1> 4.7 mM, 50% in-hibition [19]; <7> competitive vs. 5-phospho-a-d-ribose 1-diphosphate, noncompetitive vs. glutamine [24]; <2> 50% inhibition of wild-type, S283A, K305Q, R307Q and S347A mutant enzyme at 0.9 mM, 6.1 mM, 2.5 mM, 2.6 mM and 1.5 mM respectively [25]; <13> 6.12 mM, 50% inhibition [28]) [1,3,6,7,8,13,14,16,17,18,19,24,25,28] ATP <6, 8> (<6> 5 mM, 21% inhibition [13]) [1,13] CMP <6> (<6> 5 mM, 22% inhibiiton [13]) [13] GDP <1, 3, 6, 8, 13> (<3> 1 mM, approx. 75% inhibition [6]; <6> 5 mM, 40% inhibition [13]; <13> 8.5 mM, 50% inhibition [28]) [1,3,…”

“…<5> Cricetulus griseus [10] <6> Homo sapiens [8,10,13,14,29] <7> Mus musculus [10,24] <8> Columba livia (pigeon [1]) [1,2,10,12] <9> Rattus norvegicus [1,7,10] <10> Schizosaccharomyces pombe (wild type strain 972hand mutant strain ade 2hdevoid of adenylosuccinate synthase [17]) [17] <11> Glycine max (soybean, Merr. cv.…”