Regulation of C₄ Photosynthesis: Factors Affecting Cold-Mediated Inactivation and Reactivation of Pyruvate, P_I Dikinase

Abstract

The kinetics and other characteristics of cold-induced inactivation and subsequent reactivation of pyruvate, P_I dikinase from Zea mays were examined in leaf extracts and with the partially purified enzyme. Significant inactivation occurred at about 10°C and the rate of inactivation increased as the temperature was reduced below 10°C. The rate of inactivation at 0°C varied with Mg²⁺ concentration; the half-time for inactivation was about 5 min with 2 mM Mg²⁺ compared with more than 60 min with 15 mM Mg²⁺. With 10 mM Mg²⁺, used routinely, the half-time for pyruvate,P*i dikinase isolated from a variety of species varied from less than 10 min to more than 40 min. Activity was rapidly recovered by rewarming to 25°C the kinetics of reactivation were second order. Cold-induced inactivation was completely prevented by both pyruvate and phosphoenolpyruvate at concentrations of about 2 mM and 0.1 mM, respectively. The molecular weight of cold-inactivated pyruvate,P_I dikinase, assessed by mobility on Sepharose 6B, was about a quarter of the active enzyme; the tetrameric active enzyme apparently dissociates to monomers at 0°C. When illuminated leaves were exposed to lower temperature, pyruvate, P_i dikinase was partially inactivated. This activity was rapidly recovered by returning leaves to a temperature of 25°C, or by incubating extracts from the cold-treated leaves at 25°C. The dark-inactivated enzyme was apparently also modified by exposure to temperatures in the range 0-5°C.