Binding of radiolabeled N-(phosphonacetyl)-L-aspartate to aspartate transcarbamylase from Ehrlich ascites tumor cells.
Binding of N-(phosphonacetyl)-[3H]L-aspartate (PALA) to aspartate transcarbamylase (ATCase, EC 2.1.3.1) in crude extracts from Ehrlich ascites tumor cells was examined. At pH 7.4, the dissociation constant was 1.39 +/- 0.22 nM; the maximal binding capacity indicated an average intracellular ATCase concentration of 0.13 microM. The presence of phosphate, MgCl2, or CaCl2 increased the apparent dissociation constant for [3H]PALA without altering the maximal binding capacity. Phosphate, a product of the ATCase reaction, probably acts as a competitor for the PALA binding site; Mg2+ and Ca2+ may inhibit [3H]PALA binding by forming a chelate which reduces the effective concentration of the free [3H]PALA. Carbamyl phosphate was a relatively weak inhibitor of [3H]PALA binding in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer alone. Addition of NaF, an inhibitor of nonspecific phosphatases, decreased the carbamyl phosphate "Ki" as an inhibitor of [3H]PALA binding to 7 microM, a value close to the Km. NaF appears to act as an inhibitor of carbamyl phosphatase activity present in the cell extract. A first-order dissociation rate constant of 0.050 +/- 0.004 min-1 (T1/2 = 14 min) was determined by following displacement of [3H]PALA with excess unlabeled PALA. The dissociation rate was strongly temperature dependent. A second-order rate constant of 3.6 X 10(7) liters mol-1 min-1 was calculated from this rate constant and the dissociation constant. Using these kinetic constants, a simple computer model predicted that PALA binding to ATCase is 95% complete within 14 min under the conditions of the assay; at intracellular ATCase concentrations, binding is slightly faster. These results are discussed in the context of both the kinetics of inhibition and the reversal of inhibition of pyrimidine synthesis within the intact cell.
D000818:Animals; D000964:Antimetabolites, Antineoplastic / Q000378:metabolism*; D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D007700:Kinetics; D051379:Mice; D009943:Organophosphorus Compounds / Q000378:metabolism*; D010746:Phosphonoacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism*; D011485:Protein Binding; D014316:Tritium
D000964:Antimetabolites, Antineoplastic; D009943:Organophosphorus Compounds; D014316:Tritium; D001224:Aspartic Acid; C013195:sparfosic acid; D001221:Aspartate Carbamoyltransferase; D010746:Phosphonoacetic Acid