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Phosphoenolpyruvate Carboxytransphosphorylase

II. CRYSTALLIZATION AND PROPERTIES

From the ¹ From the Department of Biochemistry, Western Reserve University, School of Medicine, Cleveland, Ohio 44106

Phosphoenolpyruvate carboxytransphosphorylase catalyzes reversibly the formation of oxalacetate and pyrophosphate from phosphoenolpyruvate, bicarbonate, and orthophosphate, and also catalyzes in the absence of bicarbonate the irreversible conversion of phosphoenolpyruvate and inorganic orthophosphate to pyruvate and inorganic pyrophosphate. Conditions have been investigated which influence the yield of the enzyme from Propionibacterium shermanii. The yield is higher with glycerol than with glucose as a substrate and increases with time of fermentation, 17 to 50 days being optimum. The enzyme has been purified by ammonium phosphate or sulfate fractionation, and chromatography on cellulose-phosphate and diethylaminoethyl cellulose. The enzyme has been crystallized in the form of very small needles. The resulting enzyme is nearly homogenous on sedimentation, and the molecular weight is 430,000 ± 30,000. The electrophoretic mobility (microns) descending was 10.3 x 10^-5 cm² per volt. Treatment with 6 m urea yields a subunit or units which after dialysis have a sedimentation constant of s_20,w = 7.0 S.

The pH optimum of the forward reaction, P-enolpyruvate to oxalacetate, extends over the broad range of 6.3 to 8.0. It is somewhat narrower for the back reaction from oxalacetate to P-enolpyruvate. Mg²⁺, Mn²⁺, or Co²⁺ is required for both the forward and back reactions. The reaction is about 7 times faster from P-enolpyruvate to oxalacetate than from oxalacetate to P-enolpyruvate. The K_m values, expressed as millimolar concentration, were as follows for the forward reaction: P-enolpyruvate, 0.5; phosphate, 1.2; Mg²⁺, 1.2; Mn²⁺, 0.5; Co²⁺, 0.5; CO₂ plus HCO₃^-, 4.0 at pH 6.5 and 9.5 at pH 7.8. In the back reaction, the values were: oxalacetate, 0.47; Mg²⁺, 2.5; and pyrophosphate, 0.22.

The catalysis is inhibited by numerous compounds, including KCl and NaCl at high ionic strength. Sulfate is a noncompetitive inhibitor of the forward reaction with a K_i of about 20 mm. Buffers such as Tris-HCl, glycylglycine, and imidazole inhibit both the forward and back reactions. All the products and substrates inhibit the back reaction at quite low concentrations.

The enzyme is stable in the absence of thiols, but the reaction is stimulated by a large variety of such compounds. Ethylenediaminetetraacetate inhibits the forward reaction but the inhibition is reversible.

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