Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue.
Ribulosebisphosphate carboxylase/oxygenase is reversibly activated by the reaction of CO2 with a specific lysyl residue (Lys191 of the Rhodospirillum rubrum enzyme) to form a carbamate that coordinates an essential Mg2+ cation. Surprisingly, the Lys191----Cys mutant protein, in the presence of CO2 and Mg2+, exhibits tight binding of the reaction intermediate analogue 2-carboxyarabinitol bisphosphate [Smith, H. B., Larimer, F. W., & Hartman, F. C. (1988) Biochem. Biophys. Res. Commun. 152, 579-584], a property normally equated with effective coordination of the Mg2+ by the carbamate. Catalytic ineptness of the Cys191 mutant protein, despite its ability to coordinate Mg2+ properly, might be due to the absence of the carbamate nitrogen. To investigate this possibility, we have evaluated the ability of exogenous amines to restore catalytic activity to the mutant protein. Significantly, the Cys191 protein manifests ribulose bisphosphate dependent fixation of 14CO2 when incubated with aminomethanesulfonate but not ethanesulfonate. This novel activity reflects a Km value for ribulose bisphosphate which is not markedly perturbed relative to wild-type enzyme, a Km for Mg2+ which is in fact decreased 10-fold, and rate saturation with respect to aminomethanesulfonate (Kd = 8 mM). Chromatographic and spectrophotometric analyses reveal the product of CO2 fixation to be D-3-phosphoglycerate, while turnover of [1-3H]ribulose bisphosphate into [3H]phosphoglycolate confirms oxygenase activity. We conclude that aminomethanesulfonate restored ribulosebisphosphate carboxylase/oxygenase activities to the Cys191 mutant protein by providing a nitrogenous function which satisfies a catalytic demand normally met by the carbamate nitrogen of Lys191.
Smith
H B
HB
Biology Division, Oak Ridge National Laboratory, Tennessee.
Hartman
F C
FC
D001665:Binding Sites; D002219:Carbamates / Q000737:chemistry; D002245:Carbon Dioxide / Q000378:metabolism; D007700:Kinetics; D009154:Mutation; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D012273:Ribulose-Bisphosphate Carboxylase / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013451:Sulfonic Acids / Q000737:chemistry
D002219:Carbamates; D013451:Sulfonic Acids; D002245:Carbon Dioxide; D012273:Ribulose-Bisphosphate Carboxylase; C045904:aminomethanesulfonic acid