Simultaneous kinetic analysis of ribulose 1,5-bisphosphate carboxylase/oxygenase activities

Abstract

An assay was developed for simultaneous kinetic analysis of the activities of the bifunctional plant enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase [EC 4.1.1.39]. [1-(14)C,5-(3)H]Ribulose 1,5-bisphosphate (RuBP) was used as the labeled substrate. Tritium enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate V(c)/V(o) ratios from the expression A/(B-A) where A and B represent the (3)H/(14)C isotope ratios of doubly labeled RuBP and 3-PGA, and V(c) and V(o) represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-(14)C]glucose and [6-(3)H]glucose using the enzymes of the pentose phosphate pathway coupled with phosphoribulokinase.The kinetic properties of a commercial preparation of fully activated spinach carboxylase were studied under approximated physiological conditions of 20% O(2) (252 micromolar), 295 mul/l CO(2) (10 micromolar), 25 C, and pH 8.19. The V(c)/V(o) ratio was, within experimental error, constant at 30 seconds and 1 minute. This double label assay method may be used to calculate V(c)/V(o) ratios for the Laing-Ogren-Hageman equation, V(c)/V(o) = (V(c)K(o)/V(o)K(c)) ([CO(2)]/[O(2)]) where V(c) and V(o) represent V(max), and K(c) and K(o) represent Michaelis constants for the carboxylase and oxygenase activities, respectively.