Examination of the roles of the cysteinyl residues of spinach phosphoribulokinase by site-directed mutagenesis
- Oak Ridge National Lab., TN (United States) Univ. of Tennessee, Oak Ridge (United States)
The Calvin Cycle enzyme phosphoribulokinase (PRK) is reversibly deactivated by the formation of an intrasubunit disulfide bond between two active-site cysteines, Cys15 and Cys55. To determine whether these two residues have a role in catalysis, site-directed mutagenesis has been applied to a cDNA clone to individually replace the cysteines with serine or alanine. The remaining cysteines of PRK (Cys244 and Cys250) were similarly substituted. The mutant genes were expressed in Escherichia coli MV 1190. The specific activity of PRK in which Cys244 or Cys250 had been replaced was indistinguishable from wild-type enzyme. PRK in which Cys16 is changes to serine was about 90% as active as wild-type enzyme, while the alanine substitution was 45% as active. The Cys55 mutant proteins were more impaired, retaining only 5-15% of native activity. The K{sub m} of both ATP and ribulose 5-phosphate was determined for the C16S, C16A, C55S, and C55A enzymes. Only the K{sub m} of ribulose 5-phosphate for the position 55 mutants deviated significantly from that of wild-type enzyme, being elevated 4-fold in C55S and 8-fold in C55A PRK. These results identify Cys55 as the sole cysteine with a facilitative role in PRK catalysis and suggest that oxidative deactivation largely reflects loss of an active-site sulfhydryl rather than major conformational changes.
- DOE Contract Number:
- AC05-84OR21400
- OSTI ID:
- 5312102
- Report Number(s):
- CONF-9104107--
- Journal Information:
- FASEB Journal (Federation of American Societies for Experimental Biology); (United States), Journal Name: FASEB Journal (Federation of American Societies for Experimental Biology); (United States) Vol. 5:4; ISSN 0892-6638; ISSN FAJOE
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINO ACIDS
BACTERIA
BIOLOGICAL FUNCTIONS
CARBOXYLIC ACIDS
CATALYSIS
CYSTEINE
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
GENES
MICROORGANISMS
MOLECULAR BIOLOGY
MUTAGENESIS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PROTEINS
THIOLS
TRANSFERASES