Role of Cysteine Residues in 4-Oxalomesaconate Hydratase from Pseudomonas ochraceae NGJ1
2007
LI, Suhong | KIMURA, Maho | TAKASHIMA, Teruo | HAYASHI, Kunihiko | INOUE, Kazunori | ISHIGURO, Ryou | SUGISAKI, Hiroyuki | MARUYAMA, Kiyofumi
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 HgCl₂; and (v) only replacement of Cys-186 remarkably improved the stability of the enzyme in the absence of reducing reagent.
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