Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue
- Oak Ridge National Lab., TN (United States)
Ribulosebisphosphate carboxylase/oxygenase is reversibly activated by the reaction of Co{sub 2} with a specific lysyl residue to form a carbamate that coordinates an essential Mg{sup 2+} cation. Surprisingly, the Lys191{yields}Cys mutant protein, in the presence of Co{sub 2} and Mg{sup 2+} exhibits tight binding of the reaction intermediate analogue 2-carboxyarabinitol bisphosphate a property normally equated with effective coordination of the Mg{sup 2+} by the carbamate. Catalytic ineptness of the Cys191 mutant protein, despite its ability to coordinate Mg{sup 2+} properly, might be due to the absence of the carbamate nitrogen. To investigate this possibility, the authors 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 {sup 14}CO{sub 2} when incubated with aminomethanewsulfonate but not ethanesulfonate. This novel activity reflects a K{sub m} value for ribulose bisphosphate which is not markedly perturbed relative to wild-type enzyme, a K{sub m} for Mg{sup 2+} which is in fact decreased 10-fold, and rate saturation with respect to aminomethanesulfonate. Chromatographic and spectrophotometric analyses reveal the product of CO{sub 2} fixation to be D-3-phosphoglycerate while turnover of (1-{sup 3}H)ribulose bisphosphate into ({sup 3}H)phosphoglycolate confirms oxygenase activity. The authors 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.
- DOE Contract Number:
- AC05-84OR21400
- OSTI ID:
- 5046800
- Journal Information:
- Biochemistry; (United States), Vol. 30:21; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
RIBULOSE DIPHOSPHATE CARBOXYLASE
ENZYME ACTIVITY
BIOCHEMISTRY
CARBAMATES
CARBON 14 COMPOUNDS
CARBON DIOXIDE
CARBON DIOXIDE FIXATION
LYSINE
TRITIUM COMPOUNDS
AMINO ACIDS
CARBON COMPOUNDS
CARBON OXIDES
CARBON-CARBON LYASES
CARBONIC ACID DERIVATIVES
CARBOXY-LYASES
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
CHALCOGENIDES
CHEMISTRY
ENZYMES
HYDROGEN COMPOUNDS
LABELLED COMPOUNDS
LYASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDES
OXYGEN COMPOUNDS
550201* - Biochemistry- Tracer Techniques