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A Kinetic Characterization of Slow Inactivation of Ribulosebisphosphate Carboxylase during Catalysis

1990; Oxford University Press; Volume: 93; Issue: 4 Linguagem: Inglês

10.1104/pp.93.4.1376

1532-2548

Daryl L. Edmondson, Murray R. Badger, T. John Andrews,

Enzyme Structure and Function

The catalytic activity of ribulosebisphosphate carboxylase (Rubisco) declined as soon as catalysis was initiated by exposure to its substrate, d-ribulose-1,5-bisphosphate (ribulose-P(2)). 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 CO(2) concentration and at low pH. The inhibitors 6-phosphogluconate and H(2)O(2) alleviated the inactivation, increasing the final/initial rate ratio and the half-time. Varying ribulose-P(2) concentration in the range above that required to saturate catalysis did not affect the kinetics of inactivation. The affinities for CO(2) and ribulose-P(2) were unaffected by the inactivation. The decline in activity occurred with preparations of ribulose-P(2) which contained no detectable d-xylulose-1,5-bisphosphate and also with ribulose-P(2) 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-P(2). We conclude that the inactivation is not caused by readily-reversible binding of ribulose-P(2) at a site different from the active site and that it is unlikely to be attributable to inhibitory contaminants in ribulose-P(2) preparations.