Aspartate transcarbamoylase from Phaseolus aureus. Partial purification and properties.
1. Aspartate transcarbamoylase from 4-day-old radicles of Phaseolus aureus was purified 190-fold by (NH(4))(2)SO(4) fractionation, DEAE-cellulose and DEAE-Sephadex chromatography and Sephadex-gel filtration. The partially purified enzyme, which required P(i) for maximum stability, had an apparent molecular weight of 83000+/-5000. 2. Uridine nucleotides were found to inhibit the activity; UMP was the most potent inhibitor, followed by UDP and UTP. No other nucleotide was found to affect the enzyme, nor could UMP inhibition be overcome by adding another nucleotide. Aspartate gives a hyperbolic substrate-saturation curve, both with and without UMP. The nucleotide inhibitor is non-competitive with respect to this substrate. Carbamoyl phosphate also yields a hyperbolic substrate-saturation curve in the absence of feedback inhibitor, but when UMP is added a sigmoidal pattern results, and the inhibition is competitive with carbamoyl phosphate. 3. The degree of inhibition by UMP is not affected by p-chloromercuribenzoate, urea, mild heat pretreatment or change in pH over the range 8.5-10.5, but is affected by temperature. 4. The aspartate analogue, succinate, both activates and inhibits the reaction, depending on the concentrations of aspartate and succinate used. 5. Kinetic studies with the partially purified enzyme showed that the K(m) for carbamoyl phosphate (0.091 mm) is much lower than that for aspartate (1.7mm). A sequential reaction mechanism was inferred from product-inhibition kinetics, with carbamoyl phosphate binding to the enzyme before aspartate, and the product, carbamoylaspartate, being released ahead of P(i). Initial-velocity studies gave a set of parallel reciprocal plots, compatible with an essentially irreversible step occurring before the binding of aspartate.
D000645:Ammonium Sulfate; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D002219:Carbamates; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010710:Phosphates; D010944:Plants / Q000201:enzymology*; D013386:Succinates / Q000494:pharmacology; D013696:Temperature; D014500:Uracil Nucleotides / Q000494:pharmacology
D002219:Carbamates; D002729:Chloromercuribenzoates; D010710:Phosphates; D013386:Succinates; D014500:Uracil Nucleotides; D001221:Aspartate Carbamoyltransferase; D000645:Ammonium Sulfate