Determination of Apparent K(m) Values for Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Using the Spectrophotometric Assay of Rubisco Activity

Abstract

The spectrophotometric assay for ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) was used to determine the rate of increase in Rubisco activity over time in the presence or absence of Rubisco activase. Polynomial approximations to the raw data were used to smooth out minor fluctuations in the spectrophotometer readings, and Rubisco activase activity was expressed as nanomoles of activated Rubisco per minute. This assay was used to examine the effects of CO(2) and the inactive-Rubisco:ribulose 1,5-bisphosphate complex (ER) on the activase-catalyzed activation reaction. Double-reciprocal plots of activase activity and ER at several concentrations of CO(2) were consistent with two-substrate Michaelis-Menton kinetics, and the apparent K(m) (CO(2)) and K(m)(ER) were determined to be 53 and 2.7 micromolar, respectively. These data do not prove that ER and CO(2) are substrates for the reaction catalyzed by activase, but they may be important to our understanding of the activation process in vivo. The implications of these data and their relation to previously published data on the effects of ER and CO(2) on activase are discussed.