Cryoenzymologic studies on arginine kinase: solvent, temperature and pH effects on the overall reaction

The overall reaction catalyzed by the phosphotransferase arginine kinase was studied at normal and subzero temperatures. Ethylene glycol was used as the antifreeze and its effects on the Km values of substances, kcat and pH profiles were investigated in detail. a) The Km values for the substrate (2 mM for ATP and 0.6 mM for arginine) were little affected by the solvent composition or temperature of the reaction mixture. b) At concentration of ethylene glycol higher than 40% there was a sharp drop of enzyme activity. c) Ethylene glycol induces a large shift in the enzymic pK D) At -5 degrees C in 40% of solvent there was a break in the Arrhenius plot suggesting a change of the rate-limiting step. The relevance of these results to the reaction pathway of arginine kinase is discussed. In addition, controlled perturbations induced by cosolvent and temperature appear as useful tools for further kinetic investigations.