Simultaneous Kinetic Analysis of Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase Activities

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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]. [1-14C,5-3H]Ribulose1,5-bisphosphate (RuBP) was used as the labeled substrate. 3H enrichment of the doubly labeled 3-phosphoglycerate (3-PGA) product, common to both enzyme activities, may be used to calculate Vc/Vo ratios from the expression A/(B-A) where A and B represent the 3H/14C isotope ratios of doubly labeled RuBP and 3-PGA, and Vc and Vo represent the activities of carboxylase and oxygenase, respectively. Doubly labeled substrate was synthesized from [2-14C]glucose and [6-3H]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% O2 (252 .mu.M), 295 .mu.l/l CO2 (10 .mu.M), 25.degree. C, and pH 8.19. The Vc/Vo ratio was, within experimental error, constant at 30 s and 1 min. This double label assay method may be used to calculate Vc/Vo ratios for the Laing-Ogren-Hageman equation, Vc/Vo = (VcKo/VoKc) ([CO2]/[O2]) where Vc and Vo represent Vmax, and Kc and Ko represent Michaelis constants for the carboxylase and oxygenase activities, respectively.