The isocitrate dehydrogenase kinase phosphatase from Escherichia coli is highly sensitive to in-vitro oxidative conditions - Role of cysteine67 and cysteine108 in the formation of a disulfide-bonded homodimer

Isocitrate dehydrogenase kinase/phosphatase (IDHK/P) is a homodimeric enzym e which controls the oxidative metabolism of Escherichia coli, and exibits a high intrinsic ATPase activity. When subjected to electrophoresis under n onreducing conditions, the purified enzyme migrates partially as a dimer. T he proportion of the dimer over the monomer is greatly increased by treatme nt with cupric 1,10 phenanthrolinate or 5,5'-dithio-bis(2-nitrobenzoic acid ), and fully reversed by dithiothreitol, indicating that covalent dimerizat ion is produced by a disulfide bend. To identify the residue(s) involved in this intermolecular disulfide-bond, each of the eight cysteines of the enz yme was individually mutated into a serine. It was found that, under nonred ucing conditions, the electrophoretic patterns of all corresponding mutants are identical to that of the wild-type, except for the Cys67-->Ser which m igrates exclusively as a monomer and for the Cys108-->Ser which migrates pr eferentially as a dimer. Furthermore, in contrast to the wild-type enzyme a nd all the other mutants, the Cys67-->Ser mutant still migrates as a monome r after treatment with cupric 1,10 phenanthrolinate. This result indicates that the intermolecular disulfide bond involves only Cys67 in each IDHK/P w ild-type monomer. This was further supported by mass spectrum analysis of t he tryptic peptides derived from either the cupric 1,10 phenanthrolinate-tr eated wild-type enzyme or the native Cys108-->Ser mutant, which show that t hey both contain a Cys67-->Cys67 disulfide bond. Moreover, both the cupric 1,10 phenanthrolinate-treated wild-type enzyme and the native Cys108-->Ser mutant contain another disulfide bond between Cys356 and Cys480. Previous r esults have shown that this additional Cys356-Cys480 disulfide bond is intr amolecular [Oudot, C., Jault, J.-M., Jaquinod, M., Negre, D., Frost, J.-F, Cozzone, A.J. & Cortay, J.-C. (1998) Eur J. Biochem. 258, 579-585].