ENZYMES OF PYRUVATE AND PHOSPHOENOLPYRUVATE CARBOXYLATION IN RUMEN BACTERIA

Abstract

Extracts of mixed bacteria collected from bovine rumen fluid contained enzymes that carboxylated pyruvate and phosphoenolpyruvate (PEP). Fresh extracts showed high pyruvate carboxylase activity (EC 6.4.1.1. pyruvate carboxylase) which, however, was cold-labile and lost activity on dialysis at 4 C. Enzyme(s) catalyzing the carboxylation of PEP were stable under these conditions. The carboxylation of PEP was maximally stimulated by ADP and to a lesser degree by GDP. The ADP–requiring PEP carboxykinase [EC 4.1.1.49 phosphoenolpyruvate carboxykinase (ATP)] was equally active with either Mg++ or Mn++ and showed maximum activity at pH 6.5. The GDP–requiring PEP carboxykinase [EC 4.1.1.32 phosphoenolpyruvate carboxykinase (GTP)] required Mn++ and was almost inactive if Mg++ was substituted. Maximum activity was at pH 7.0. These nucleotides were most effective at 2.5-mM concentration and were inhibitory at higher concentrations. In the absence of added ADP or GDP, the carboxylation of PEP continued at a low but persistent rate. Precipitation with ammonium sulphate and adsorption on calcium phosphate gel resulted in fractions containing different proportions of the three activities. These results suggest that in mixed rumen bacterial extracts there are four separate enzymes capable of synthesizing oxaloacetate: one that catalyzes the carboxylation of pyruvate and three that catalyze the carboxylation of PEP.