[Reactivity and function of cysteine residues in imidase from Pseudomonas putida YZ-26]

We investigated the reactivity and function of two cysteine residues in imidase (CIH) by site-directed mutagenesis.

Three variants of imidase (CIH) were constructed with Cys7 and Cys108 or only one of them substituted with Gly. The two thiol groups of Cys7 and Cys108 of imidase were specifically modified separately or collectively by dithio-bis-nitrobenzoic acid (DTNB) in the native state. It was also confirmed by SDS-PAGE analysis. To further verify the above results, the oligomeric structure and sulfhedryl groups of native and mutant CIH were also examined by measuring zinc binding ability and molecular size under different concentrations of H2O2.

Compared with CIH, CIH108 retained 72% activity, while CIH7, 108 and CIH7 had no activity using DL-hydantoin as substrate. The spectral detection result shown that the two thiol groups were both in a free state. It is indicated that CIH and CIH108 are tetramer, CIH7,108 is multimer, and CIH7 is a mixture of monomer and multimer. The zinc binding ability of CIH108 was still relative high, while CIH7 and CIH7,108 decreased obviously. The increased concentration of H2O2 could increase the intrachain disulfide bond of CIH and the interchain disulfide bond of CIH108.

All data imply that the Cys7 is required for binding zinc ion and maintaining the stable structure of enzymatic molecule.