A novel method for determining equilibrium constants. CTP:phosphorylcholine cytidyltransferase.

A novel method for the determination of equilibrium constants for reversible reactions is described. The method is based on the measurement of initial velocities of isotope transfer for a given substrate-product pair of both the forward and reverse reactions as a function of the mass action of reactants. The reciprocal values of these initial velocities are plotted against the mass action ratios of reactants. The observed Keq is the abscissa of the intersection point of these reciprocal plots, i.e. the mass action ratio at which the initial velocities of isotope transfer for both the forward and reverse reaction are identical, that is, when isotope exchange is occurring. In this manner, an observed Keq of 0.2 was obtained from CTP:phosphorylcholine cytidyltransferase (CTP:cholinephosphate cytidyltransferase, EC 2.7.7.15) at 37 degrees C and pH 7.5 under physiological conditions 1.0 mM free Mg2+ and 0.15 M salt concentration. A comparison of this value with the in vivo mass action of reactants calculated from published data indicates that this reaction is rate-limiting in the rat liver (Infante, J.P. (1977) Biochem. J. 167, 847--849).