A Kinetic Characterization of Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis

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Abstract
The catalytic activity of ribulosebisphosphate carboxylase (Rubisco) [from Spinacia oleracea] declined as soon as catalysis was initiated by exposure to its substrate, D-ribulose-1,5-bisphosphate (ribulose-P2). The decline continued exponentially, with a half-time of approximately 7 minutes until, eventually, a steady state level of activity was reached which could be as low as 15% of the initial activity. The ratio of the steady state activity to the initial activity was lower at low CO2 concentration and at low pH. The inhibitors 6-phosphogluconate and H2O2 alleviated the inactivation, increasing the final/initial rate ratio and the half-time. Varying ribulose-P2 concentration in the range above that required to saturate catalysis did not affect the kinetics of inactivation. The affinities for CO2 and ribulose-P2 were unaffected by the inactivation. The decline in activity occurred with preparations of ribulose-P2 which contained no detectable D-xylulose-1,5-bisphosphate and also with ribulose-P2 which had been generated enzymatically immediately before use. Inclusion of an aldolase system for removing D-xylulose-1,5-bisphosphate also did not alter the inactivation process. The inactivated Rubisco did not recover after complete exhaustion of ribulose-P2. We conclude that the inactivation is not caused by readily-reversible binding of ribulose-P2 at a site different from the active site and that is unlikely to be attributable to inhibitory contaminants in ribulose-P2 preparations.

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