Purification and properties of mammalian liver cytidine triphosphate synthetase.

Cytidine triphosphate synthetase detected earlier in crude rat liver extracts by other workers (HURLBERT, R. B., AND KAMMEN, H. O., J. Biol. Chem., 235, 443 (1960)) has been purified about 350-fold from rat and calf liver. The enzyme catalyzes the conversion of UTP to CTP in the presence of ATP, L-glutamine, Mg++ , and a sulfhydryl-containing compound. Although it is not an absolute requirement for the reaction, GTP is necessary for optimal activity. The kinetics of the reaction have been investigated. The velocity as a function of UTP concentration at saturating and nonsaturating concentrations of either ATP or GTP strongly resembled Michaelis-Menten hyperbolas. Similarly, hyperbolic relationships were found between the velocity and the concentration of L-glutamine either in the absence or presence of GTP. All Hill plots of these data were linear and gave Hill coefficients of unity. Thus, neither positive nor negative cooperative interactions between substrate sites or activator sites were detectable with this enzyme. These results sharply differentiate the liver enzyme from the analogous CTP synthetase of Escherichia coli B studied by other workers (LONG, C. W., AND PARDEE, A. B., J. Biol. Chem., 242, 4715 (1967)). The bacterial enzyme has a much more stringent requirement for GTP but has no need for a sulfhydryl compound. The bacterial enzyme can exhibit both strong positive and negative cooperative substrate site interactions. With regard to regulation of enzymatic activity at the molecular level, it is noteworthy that liver and E. coli differ fundamentally at another locus in the biosynthetic pathway de nouo for pyrimidine nucleotides. The liver aspartate transcarbamylase exhibits Michaelis-Menten behavior and the bacterial enzyme, sigmoidal kinetics (GERHART, J. C., AND PARDEE, A. B., J. Biol. Chem., 237, 891 (1962)). The liver enzyme but not the bacterial enzyme is insensitive to negative feedback by CTP and to activation by ATP (BRESNICK, E., AND MOSSE, H., Biochem. J., 101, 63 (1966)).