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 immobilizes the flexible loop 6 of the beta/alpha barrel domain in its closed 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 implicated Lys329 in the addition of CO2 to the initial enediol(ate) intermediate: mutants at position 329 catalyze enolization of ribulose 1,5-bisphosphate 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-bisphosphate. Using the chemical rescue method of Toney and Kirsch [(1989) Science 243, 1485-1488], we show that these defects are partially overcome by exogenous amines. For example, ethylamine enhances the carboxylation rate of K329A by about 80-fold and strengthens complexation of 2-carboxyarabinitol 1,5-bisphosphate. The CO2/O2 specificity of K329A 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 Brønsted relationship with 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 K329C suggest that the amines bind to the enzyme in the position voided by the mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
Harpel
M R
MR
Protein Engineering Program, Oak Ridge National Laboratory, Tennessee 37831-8077.
Hartman
F C
FC
D000588:Amines / Q000737:chemistry*; D001665:Binding Sites; D007700:Kinetics; D008239:Lysine / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D012247:Rhodospirillum rubrum / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry* / Q000235:genetics
D000588:Amines; D012273:Ribulose-Bisphosphate Carboxylase; D008239:Lysine