CHEMICAL RESCUE BY EXOGENOUS AMINES OF A SITE-DIRECTED MUTANT OF RIBULOSE 1,5-BISPHOSPHATE CARBOXYLASE OXYGENASE THAT LACKS A KEY LYSYL RESIDUE/

Ligand binding to ribulose 1,5-bisphosphate carboxylase/oxygenase immo bilizes the flexible loop 6 of the beta/alpha barrel domain in its clo sed conformation. Lys329, located at the apex of this loop, interacts electrostatically with Glu48 of the adjacent subunit and with the CO2- derived carboxylate of the carboxylated reaction intermediate [Knight et al. (1990) J. Mol. Biol. 215, 113-160]. Previous studies have impli cated Lys329 in the addition of CO2 to the initial enediol(ate) interm ediate: mutants at position 329 catalyze enolization of ribulose 1,5-b isphosphate and processing of isolated carboxyketone intermediate, but are severely impaired in overall carboxylation and the tight-binding of the carboxylated intermediate analogue 2-carboxyarabinitol 1,5-bisp hosphate. Using the chemical rescue method of Toney and Kirsch [(1989) Science 243, 1485-1488], we show that these defects are partially ove rcome by exogenous amines. For example, ethylamine enhances the carbox ylation rate of K329A by about 80-fold and strengthens complexation of 2-carboxyarabinitol 1,5-bisphosphate. The CO2/O-2 specificity of K329 A is increased by amines, but remains lower than the wild-type value. Despite the pronounced enhancement of carboxylase activity, amines do not influence the rate at which ribulose 1,5-bisphosphate is enolized by K329A. Rescue of K329A follows an apparent Bronsted relationship wi th a beta of 1, implying complete protonation of amine in the rescued transition state. Rate saturation with respect to amine concentration and the different steric preferences for amines between K329A and K329 C suggest that the amines bind to the enzyme in the position voided by the mutation. Two side products, one formed via beta-elimination of p hosphate from enediol(ate) intermediate and the other linked preferent ially to the oxygenase pathway, predominate in the absence of amines. Amines suppress these abortive products in favor of normal turnover pr oducts. Collectively, these results not only verify that Lys329 functi ons primarily in the gaseous substrate addition to the enediol(ate) bu t also demonstrate the residue's crucial role in stabilizing reaction intermediates.