Deoxycytidine Kinase: III. KINETICS AND ALLOSTERIC REGULATION OF THE CALF THYMUS ENZYME

Abstract

Abstract The kinetic behavior of calf thymus deoxycytidine kinase was studied over a 100-fold variation in deoxycytidine concentration, and several abrupt transitions were observed in slopes of double reciprocal plots, suggesting negative homotropic cooperativity in the binding of this substrate. This is in apparent contrast to the simpler kinetics seen when an alternate substrate, deoxyguanosine, or the substrate analogue, 1-β-d-arabinofuranozylcytosine, was used in place of deoxycytidine. Convex reciprocal plots were also obtained with variable ATP-Mg2- or with an alternative phosphate donor, dTTP-Mg2-, although the latter yielded more marked negative curvature and lower Vmax values. Inhibition by end product, dCTP, was complex, particularly when deoxycytidine was varied over a wide range of concentrations, but below the first transition concentration dCTP plotted as a noncompetitive inhibitor. With variable ATP-Mg2-, dCTP gave the appearance of noncompetitive inhibition at low ATP-Mg2-, but shifted to apparent competitive inhibition against high phosphate donor concentrations. End product inhibition is overcome by dTTP at low deoxycytidine concentrations, but with increased nucleoside concentrations dTTP replaces the dCTP effect with its own highly complex pattern of inhibition. Like dTTP, UDP also stimulates at low nucleoside concentrations and becomes inhibitory as the deoxycytidine concentration is elevated. However, this inhibition can be reversed by dTTP, provided that the ATP-Mg2- concentration is limiting.