Properties of Phosphoenolpyruvate Carboxykinase Operative in C₄ Pathway Photosynthesis

Abstract

A procedure is described for partially purifying phosphoenolpyruvate carboxykinase [ATP : oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] from leaves of Chloris gayana Kunth. In three steps the enzyme was purified about 60-fold with 22% recovery of activity. This procedure removes enzymes, particularly malate dehydrogenase, that preclude the use of a simple spectrophotometric assay for phosphoenolpyruvate carboxykinase. The activity of the enzyme in the direction of oxaloacetate decarboxylation was about 10 times that in the reverse direction. At the optimal pH of 8.0, ATP was the preferred nucleoside triphosphate but CTP, UTP, GTP and ITP were also active. A requirement for Mn²⁺ could not be replaced by Mg²⁺. The Michaelis constants for oxaloacetate and ATP were 0.035 mM and 0.024 nM, respectively. The photosynthetic intermediates fructose 1,6-bisphosphate, 3-phosphoglyceric acid and dihydroxyacetone phosphate significantly inhibited the enzyme at concentrations in the region of 1-5 mM. Unlike the phosphoenolpyruvate carboxykinase from other sources, the capacity of the leaf enzyme to catalyse the decarboxylation of oxaloacetate to pyruvate was negligible. The properties of the enzyme are discussed in relation to its proposed role in C₄ pathway photosynthesis.