A LOW TEMPERATURE-INDUCED REVERSIBLE TRANSITION BETWEEN DIFFERENT KINETIC FORMS OF MAIZE LEAF PHOSPHOENOLPYRUVATE CARBOXYLASE

Partially purified (4-6 fold) maize (Zea mays) leaf phosphoenolpyruvate (PEP) carboxylase (PEPC) was unstable when incubated in the cold (0-4-degrees-C). The cold-inactivated enzyme could regain some or most of its activity during the time-courses of assays, with a half-time of activation on the order of minutes. The lags could be resolved into protein-dependent and protein-independent components, which are suggested to be due to protein association and to conformational change(s), respectively. Incubation at low temperature caused major reversible changes in the kinetic properties of PEPC. Depending on assay pH, at a fixed concentration of free magnesium, the kinetics of initial rates of the cold-incubated enzyme displayed either no cooperativity with respect to Mg-PEP (pH 7.1) or negative cooperativity (pH 7.8). Addition of glucose-6-phosphate (G6P) to assays at pH 7.1 changed the kinetics to negative cooperativity. The negative cooperativity phenomenon yielded two K(m) and two V(max) values at each assay pH (K(m) of 1 and 20 mM at pH 7.1, and K(m) of 1 and 6 mM at pH 7.8). Kinetics of steady-state rates of cold-incubated PEPC were in most respects similar to those of the enzyme kept at 21-degrees-C, with positive cooperativity at pH 7.1 (K(m) of 1-2 mM or 7 mM with or without G6P, respectively) and no cooperativity at pH 7.8 (K(m) of 0.3 or 1 mM with or without G6P, respectively). At each assay pH, G6P considerably increased V(max) of steady-state rates of cold-treated enzyme, while it had no effect on V(max) of the enzyme kept at 21-degrees-C. It is suggested that incubation of maize PEPC in the cold leads to the formation of a high K(m) form of the enzyme. Exposure to assay conditions causes a slow reversal of this process towards a low K(m) form, with both forms (under certain assay conditions) having similar V(max) values. The possible basis for the reversible transition between different kinetic forms of PEPC is discussed.