Structure and function of chloroplast proteins: V. Homotropic effect of bicarbonate in RuDP carboxylase reaction and the mechanism of activation by magnesium ions

Abstract

Kinetic analyses of the CO 2-fixation reaction catalyzed by RuDP-carboxylase prepared from spinach leaves showed that the reaction rate ( 14CO 2-fixation) vs. NaHCO 3 concentrations curve was deviated from the Michaelian type. From the analysis of data by the empirical Hill equation, the interaction coefficient, n∗, was calculated to be approximately 2, indicating a homotropic interaction of the NaHCO 3 molecule. It was thus inferred that NaHCO 3 molecules interact mutually between the multiple substrate-binding sites on the enzyme molecule. Activation of the carboxylation reaction by Mg ++ was studied, and both Km (NaHCO 3) and Vmax values were found to be affected by increasing Mg ++ concentrations. The Km value for NaHCO 3 was 2 × 10 −2 m in the absence of Mg ++ and 5.6 × 10 −3 m at 2 × 10 −2 m Mg ++. However, it was found that n∗ numbers do not change by increasing Mg ++ concentrations. This feature is in sharp contrast to the case of some other regulatory enzymes. Cooperativity of the enzyme molecule with NaHCO 3 was determined at different pH values, and the higher pH value the greater cooperativity ( n∗ numbers 2) and smaller the Km value to Mg ++. The nature of Mg ++ activation was further studied as regards (1) the shift of optimum pH values of the enzyme; pH 7.5 at 10 −3 m MgCl 2 and pH 6.5 at 10 −2 m MgCl 2, and (2) the protective effect of Mg ++ association to the enzyme molecule against the subsequent attack by the proteolytic enzyme (Nagarse). Some physiological implications have been made concerning the regulatory nature of RuDP carboxylase.