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The Enzymatic Carboxylation of Phosphoenolpyruvate

III. INVESTIGATION OF THE KINETICS AND MECHANISM OF THE MITOCHONDRIAL PHOSPHOENOLPYRUVATE CARBOXYKINASE-CATALYZED REACTION

From the ¹ From the Department of Biochemistry, New York University School of Medicine, New York, New York 10016

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 ß-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 (K_m and V values) for all substrates and cofactors of the carboxykinase-catalyzed carboxylation, decarboxylation, and oxalacetate-H¹⁴CO₃^- exchange reaction were determined. The K_m 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 ß-carboxyl site on the enzyme. The K_m 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.

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