The enzymatic activity and inhibition of adenosine 5'-triphosphate-creatine transphosphorylase.

From our earlier rate measurements (l), it was not possible to develop a satisfactory kinetic scheme accounting for the dependence of reaction rate on the concentration of certain ions, such as Mg++ and Hf. Recently, however, we have found (2) that some of the anions which we were inadvertently introducing in the earlier work (e.g. in varying the concentration of Mg++ with the use of magnesium sulfate) strongly interact with this enzyme system, thereby confusing attempts at kinetic interpretation. Having recognized this effect, we have turned to the use of comparatively inert anions (e.g. acetate, glycinate), and have found that the simpler results thus obtained are well described by a plausible kinetic scheme. Our conclusions are that (with respect to either substrate) the system follows “Michaelis-Menten kinetics,” that MgATP2is probably the true substrate, that I-L4TP3is a competitive inhibitor, and that the curve relating steady state velocity to pH resembles the titration of a single ionizing group with a pK 6.5. The activity of the enzyme preparations used in the present work has been about 40% higher than the activity of crystalline preparations used in our earlier work (1). Reagents-Salts used for inhibition studies were reagent grade and twice crystallized from aqueous solution. Glycine, histidine (free base), and creatine were Cfp grade (California Corporation for Biochemical Research). Nucleotides were the highest grade available from Sigma Chemical Company. Enzymatic Assay-The initial rate of enzymatic activity at 30” was measured, as previously reported (I), by plotting the time course of formation of creatine phosphate in a series of five aliquots of the incubation mixture pipetted into a mixture of acid-molybdate. The maximal percentage of total ATP reacting was generally under 10% and the plotted initial rates were linear. Inorganic anions in the incubation mixture were avoided insofar as was possible. Sodium hydroxide was used to adjust the pH of buffer solutions, and a stock 1 M magnesium acetate solution prepared from MgAcs.4Hz0, analytical reagent grade, was sta.ndardized by precipitating magnesium hydroxyquinolate.