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Title: Restoration of enzyme activity to the Cys-329 mutant of ribulose biphosphate carboxylase/oxygenase by aminoethylation

Abstract

To examine the function of active-site Lys-329 of Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase, the residue has been replaced with other amino acids by site-directed mutagenesis. Although all substitutions results in abolishment of catalytic activity, the Cys-329 mutant provides an opportunity for determining the consequence of a subtle change in the active-site microenvironment - replacement of a lysyl ..gamma..-methylene group by a sulfur atom. Treatment of the catalytically inactive mutant protein with bromoethylamine results in 25% restoration of carboxylase activity, presumably reflecting selective aminoethylation of Cys-329. The K/sub m/ values displayed by the novel carboxylase for both CO/sub 2/ and ribulose bisphosphate are similar to those of the wild-type enzyme. Like the latter, the aminoethylated protein forms a stable quaternary complex with a transition-state analog, CO/sub 2/, and Mg/sup 2 +/. Preliminary assays indicate the ratio of carboxylase/oxygenase activity is unperturbed by the tailored structural change of the catalytic site.

Authors:
;
Publication Date:
Research Org.:
Oak Ridge National Lab., TN
OSTI Identifier:
6327130
Report Number(s):
CONF-870644-
Journal ID: CODEN: FEPRA
Resource Type:
Conference
Journal Name:
Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)
Additional Journal Information:
Journal Volume: 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CARBOXYLASE; ENZYME ACTIVITY; CYSTEINE; BIOLOGICAL FUNCTIONS; QUANTITY RATIO; OXYGENASES; AMINO ACIDS; MUTANTS; CARBON-CARBON LYASES; CARBOXY-LYASES; CARBOXYLIC ACIDS; ENZYMES; FUNCTIONS; LYASES; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; OXIDOREDUCTASES; THIOLS; 550200* - Biochemistry

Citation Formats

Smith, H B, and Hartman, F C. Restoration of enzyme activity to the Cys-329 mutant of ribulose biphosphate carboxylase/oxygenase by aminoethylation. United States: N. p., 1987. Web.
Smith, H B, & Hartman, F C. Restoration of enzyme activity to the Cys-329 mutant of ribulose biphosphate carboxylase/oxygenase by aminoethylation. United States.
Smith, H B, and Hartman, F C. Fri . "Restoration of enzyme activity to the Cys-329 mutant of ribulose biphosphate carboxylase/oxygenase by aminoethylation". United States.
@article{osti_6327130,
title = {Restoration of enzyme activity to the Cys-329 mutant of ribulose biphosphate carboxylase/oxygenase by aminoethylation},
author = {Smith, H B and Hartman, F C},
abstractNote = {To examine the function of active-site Lys-329 of Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase, the residue has been replaced with other amino acids by site-directed mutagenesis. Although all substitutions results in abolishment of catalytic activity, the Cys-329 mutant provides an opportunity for determining the consequence of a subtle change in the active-site microenvironment - replacement of a lysyl ..gamma..-methylene group by a sulfur atom. Treatment of the catalytically inactive mutant protein with bromoethylamine results in 25% restoration of carboxylase activity, presumably reflecting selective aminoethylation of Cys-329. The K/sub m/ values displayed by the novel carboxylase for both CO/sub 2/ and ribulose bisphosphate are similar to those of the wild-type enzyme. Like the latter, the aminoethylated protein forms a stable quaternary complex with a transition-state analog, CO/sub 2/, and Mg/sup 2 +/. Preliminary assays indicate the ratio of carboxylase/oxygenase activity is unperturbed by the tailored structural change of the catalytic site.},
doi = {},
journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 46:6,
place = {United States},
year = {1987},
month = {5}
}

Conference:
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