The Enzymatic Carboxylation of Phosphoenolpyruvate III. INVESTIGATION OF THE KINETICS AND MECHANISM OF THE MITOCHONDRIAL PHOSPHO-

Comparative kinetic studies at pH 6.8, 7.3, and 8.0 reveal that the phosphoenolpyruvate carboxykinase-catalyzed inosine trlphosphate-dependent oxalacetate-H14C0sexchange is much more rapid than either the over-all decarboxylation or carboxylation reactions. At pH 6.8, the relative carboxylation, decarboxylation, and oxalacetate-H14C03exchange 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-H14COSexchange reaction differ markedly from that for the decarboxylation reaction. Under conditions which permit rapid P-enolpyruvate carboxykinasecatalyzed oxalacetate-H14C0,exchange; neither GTP-GDP8J4C-, ITP-3* P-enolpyruvate-, nor oxalacetate-P-enolpyruruvate-l-%-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 oxalacetateH14C01exchange reaction or (b) the dissociation of either enzyme-P-enolpyruvate or enzyme-IDP (or GDP) as a ratelimiting step in the over-all decarboxylation reaction. The results support a two-step reaction sequence in which an ITP-dependent cleavage of the /3-carboxyl carbon-carbon bond of oxalacetate yields HCOI(or CO3 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,,, and V values) for all substrates and cofactors of the carboxykinase-catalyzed carboxylation, decarboxylation, and oxalacetate-H14COSexchange reaction were determined. The K,,, values (0.025 M and 0.027 M, respectively) for bicarbonate in the carboxylation (pH 7.5) and oxalacetate-H14COSexchange (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 HCOIis also evident in the oxalacetate-H14C03exchange reaction suggesting