PHOSPHOENOLPYRUVATE CARBOXYTRANSPHOSPHORYLASE .3. COMPARISON OF FIXATION OF CARBON DIOXIDE AND CONVERSION OF PHOSPHOENOLPYRUVATE AND PHOSPHATE INTO PYRUVATE AND PYROPHOSPHATE

Carboxytransphosphorylase from propionibacteria catalyzes the conversion of phosphoenolpyruvate, PI, and CO2 into oxalacetate and PPi and when CO2 is excluded the products are pyruvate and PPi. The latter reaction has not been studied previously. The stoichiometry of the pyruvate reaction has been determined and evidence is presented that both reactions are catalyzed by the same enzyme. A comparison of the characteristics of the two reactions is presented. The CO2 fixation reaction is reversible but the pyruvate reaction is experimentally irreversible. Both reactions have about the same pH optimum. A divalent metal is required for either reaction and Mg2+, Co2+, or Mn2+ meets this requirement. The apparent Km. values for the pyruvate reaction are Mg2+, 6.3 X 10-4 M; CO2+, 2.3 X lO-4 M; Mn2+, 6.3 X 10-4M, phosphoenolpyruvate, 3.6 X 10-5 M; and Pi, 6.6 X 10-4 M. These values are quite similar to those previously observed for the CO2 fixation reaction except for that of phosphoenolpyruvate and Mn2+ which are about tenfold lower for the pyruvate reaction. It has been shown that the presence of bicarbonate alters the apparent Km for phosphoenolpyruvate in the pyruvate reaction to a value approaching that found for the fixation of CO2. Both the pyruvate reaction and the CO2 fixation reaction are inhibited by 10-5 M EDTA even in the presence of 12 mM Mg2+ and also by other metal chelators. It is postulated that two types of metals are required: type I which is disso ciable (i.e., Mg2+, Co2+, or Mn2+) and an unidentified metal, type II, which is firmly bound to the carboxytransphosphorylase and whose function is blocked by the chelators. Co2+ removes the EDTA inhibition of the oxalacetate reaction and Cu2+ of the pyruvate reaction. This difference in the two reactions is believed to arise because in addition to type I and type II metals the pyruvate reaction requires a heavy metal. The Co2+ complexes the EDTA and thus removes it from the type II metal-restoring activity for CO2 fixation. The Cu2+ removes the EDTA from the type II metal and also serves as the heavy metal thus restoring the pyruvate reaction. Cu2+ inhibits the CO2 fixation reaction. Thiols such as mercaptoethanol stimulate the rate of the oxalacetate reaction but inhibit the pyruvate reaction. The thiols may act by complexing the heavy metals and thereby stimulate the CO2 fixation reaction but inhibit the pyruvate reaction. Further evidence of this role of thiols is the fact that the EDTA-treated enzyme is fully active in the oxalacetate reaction in the presence of Co2+ but without addition of thiol. It thus appears that different forms of the enzyme catalyze the two reactions. The pyruvate reaction apparently is not required for the conversion of phosphoenolpyruvate into pyruvate in propionibacteria since they contain pyruvate kinase. It seems likely that the physiological role of carboxytransphosphorylase is for fixation of CO2 to yield oxalacetate rather than for formation of pyruvate. © 1969, American Chemical Society. All rights reserved.