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Title: Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue

Abstract

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 thatmore » 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.« less

Authors:
;  [1]
  1. Oak Ridge National Lab., TN (United States)
Publication Date:
OSTI Identifier:
5046800
DOE Contract Number:  
AC05-84OR21400
Resource Type:
Journal Article
Journal Name:
Biochemistry; (United States)
Additional Journal Information:
Journal Volume: 30:21; Journal ID: ISSN 0006-2960
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 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

Citation Formats

Smith, H B, and Hartman, F C. Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue. United States: N. p., 1991. Web. doi:10.1021/bi00235a009.
Smith, H B, & Hartman, F C. Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue. United States. doi:10.1021/bi00235a009.
Smith, H B, and Hartman, F C. Tue . "Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue". United States. doi:10.1021/bi00235a009.
@article{osti_5046800,
title = {Demonstration of a functional requirement for the carbamate nitrogen of ribulosebisphosphate carboxylase/oxygenase by chemical rescue},
author = {Smith, H B and Hartman, F C},
abstractNote = {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.},
doi = {10.1021/bi00235a009},
journal = {Biochemistry; (United States)},
issn = {0006-2960},
number = ,
volume = 30:21,
place = {United States},
year = {1991},
month = {5}
}