The Enzymatic Carboxylation of Phosphoenolpyruvate: III. INVESTIGATION OF THE KINETICS AND MECHANISM OF THE MITOCHONDRIAL PHOSPHOENOLPYRUVATE CARBOXYKINASE-CATALYZED REACTION

Abstract

Abstract Comparative kinetic studies at pH 6.8, 7.3, and 8.0 reveal that the phosphoenolpyruvate carboxykinase-catalyzed inosine triphosphate-dependent oxalacetate-H14CO3- exchange is much more rapid than either the over-all decarboxylation or carboxylation reactions. At pH 6.8, the relative carboxylation, decarboxylation, and oxalacetate-H14CO3- exchange rates are 1.0, 8.3, and 30, respectively. While the pH optima for all three reactions are essentially the same (pH 6.6), the shapes of pH optimum curves for the carboxylation and oxalacetate-H14CO3- exchange reaction differ markedly from that for the decarboxylation reaction. Under conditions which permit rapid P-enolpyruvate carboxykinase-catalyzed oxalacetate-H14CO3- exchange; neither GTP-GDP-8-14C-, ITP-32 P-enolpyruvate-, nor oxalacetate-P-enolpyruvate-1-14C-exchange occurs at a significant rate. The inability of carboxykinase to catalyze these exchange reactions renders unlikely (a) the formation of phosphorylenzyme from GTP (or ITP) and enzyme as a step in the oxalacetate-H14CO3- exchange reaction or (b) the dissociation of either enzyme-P-enolpyruvate or enzyme-IDP (or GDP) as a rate-limiting step in the over-all decarboxylation reaction. The results support a two-step reaction sequence in which an ITP-dependent cleavage of the β-carboxyl carbon—carbon bond of oxalacetate yields HCO3- (or CO2) and an intermediate containing the elements of ITP and a 3 carbon moiety derived from carbon atoms 1, 2, and 3 of oxalacetate. Two mechanisms compatible with these results and other known characteristics of the reaction are proposed. Kinetic constants (Km and V values) for all substrates and cofactors of the carboxykinase-catalyzed carboxylation, decarboxylation, and oxalacetate-H14CO3- exchange reaction were determined. The Km values (0.025 m and 0.027 m, respectively) for bicarbonate in the carboxylation (pH 7.5) and oxalacetate-H14CO3- exchange (pH 7.3) reactions were in close agreement. Bicarbonate is a competitive inhibitor (with respect to oxalacetate) of the decarboxylation reaction. Competition between oxalacetate and HCO3- is also evident in the oxalacetate-H14CO3- exchange reaction suggesting a common HCO3- and β-carboxyl site on the enzyme. The Km values (1.6 to 5.8 x 10-4 m, pH 7.3) for ITP and GTP in the decarboxylation and oxalacetate-H14CO3- exchange reaction determined under similar conditions agreed closely. The oxalacetate-H14CO3- exchange reaction is strongly inhibited by P-enolpyruvate, whereas, the decarboxylation rate is unaffected at similar P-enolpyruvate concentrations. Both the oxalacetate-H14CO3- exchange and decarboxylation reactions are inhibited by GMP and GDP.