The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum.
In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme. Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A. The mutant Thermoplasma citrate synthase is very similar to wild-type citrate synthase in its substrate and co-factor specificities, pH profile and thermal stability. The mutation, however, has decreased the enzyme activity. The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity.
Kocabiyik
S
S
Department of Biological Sciences, Middle East Technical University, Ankara, Turkey.
Erduran
I
I
Russel
R J
RJ
Danson
M J
MJ
Hough
D W
DW
D002950:Citrate (si)-Synthase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine; D004795:Enzyme Stability; D004926:Escherichia coli; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics; D013822:Thermoplasma / Q000201:enzymology*
D011994:Recombinant Proteins; D002950:Citrate (si)-Synthase; D003545:Cysteine