Role of cysteine residues in 4-oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1.
4-Oxalomesaconate hydratase from Pseudomonas ochraceae NGJ1 is unstable in the absence of reducing reagents such as dithiothreitol, and strongly inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). To study the role of cysteine residues in enzyme catalysis, the eight individual cysteine residues of the enzyme were replaced with serine residues by site-directed mutagenesis. The catalytic properties and chemical modification of wild- and mutant type-enzymes by DTNB showed that (i) none of eight cysteine residues was essential for enzyme catalysis; (ii) the inhibition by DTNB was mostly due to modification of Cys-186; (iii) Cys-96 might be another residue reacting with DTNB, and its modification caused an increase in the K(m)-value for 4-oxalomesaconate; (iv) the other six cysteine residues were inaccessible to DTNB, but susceptible to HgCl(2); and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.
Li
Suhong
S
Department of Biomolecular Science, Faculty of Engineering, Gifu University, Gifu, Japan.
Kimura
Maho
M
Takashima
Teruo
T
Hayashi
Kunihiko
K
Inoue
Kazunori
K
Ishiguro
Ryou
R
Sugisaki
Hiroyuki
H
Maruyama
Kiyofumi
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003080:Cold Temperature; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics; D004269:DNA, Bacterial / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D006836:Hydro-Lyases / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011549:Pseudomonas / Q000201:enzymology*; D012241:Rhodopseudomonas / Q000737:chemistry; D013439:Sulfhydryl Reagents / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D006836:Hydro-Lyases; C045057:ligJ protein, Pseudomonas ochraceae; D015032:Zinc; D003545:Cysteine