In situ behavior of the pyrimidine pathway enzymes in Saccharomyces cerevisiae. I. Catalytic and regulatory properties of aspartate transcarbamylase.
A permeabilization procedure was adapted to allow the in situ determination of aspartate transcarbamylase activity in Saccharomyces cerevisiae. Permeabilization is obtained by treating cell suspensions with small amounts of 10% toluene in absolute ethanol. After washing, the cells can be used directly in the enzyme assays. Kinetic studies of aspartate transcarbamylase (EC 2.1.3.2) in such permeabilized cells showed that apparent Km for substrates and Ki for the feedback inhibitor UTP were only slightly different from those reported using partially purified enzyme. The aspartate saturation curve is hyperbolic both in the presence and absence of UTP. The inhibition by this nucleotide is noncompetitive with respect to aspartate, decreasing both the affinity for this substrate and the maximal velocity of the reaction. The saturation curves for both substrates give parallel double reciprocal plots. The inhibition by the products is linear noncompetitive. Succinate, an aspartate analog, provokes competitive and uncompetitive inhibitions toward aspartate and carbamyl phosphate, respectively. The inhibition by phosphonacetate, a carbamyl phosphate analog, is uncompetitive and noncompetitive toward carbamyl phosphate and aspartate, respectively, but pyrophosphate inhibition is competitive toward carbamyl phosphate and noncompetitive toward aspartate. These results, as well as the effect of the transition state analog N-phosphonacetyl-L-aspartate, all exclude a random mechanism for aspartate transcarbamylase. Most of the data suggest an ordered mechanism except the substrates saturation curves, which are indicative of a ping-pong mechanism. Such a discrepancy might be related to some channeling of carbamyl phosphate between carbamyl phosphate synthetase and aspartate transcarbamylase catalytic sites.
D001221:Aspartate Carbamoyltransferase / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D002250:Carbon Radioisotopes; D005246:Feedback; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011743:Pyrimidines / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014544:Uridine Triphosphate / Q000494:pharmacology
D002250:Carbon Radioisotopes; D011743:Pyrimidines; D001224:Aspartic Acid; D001221:Aspartate Carbamoyltransferase; D014544:Uridine Triphosphate