| Summary: | The strictly conserved αSer162 residue in the Co-type nitrile hydratase from <i>Pseudonocardia thermophila</i> JCM 3095 (<i>Pt</i>NHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein species: one was the apoform (αSer<sup>A</sup>) that exhibited no observable activity, and the second (αSer<sup>B</sup>) contained its full complement of cobalt ions and was active with a <i>k</i><sub>cat</sub> value of 63 ± 3 s<sup>−1</sup> towards acrylonitrile at pH 7.5. The X-ray crystal structure of αSer<sup>A</sup> was determined at 1.85 Å resolution and contained no detectable cobalt per α<sub>2</sub>β<sub>2</sub> heterotetramer. The axial αCys108 ligand itself was also mutated into Ser, Met, and His ligands. All three of these αCys108 mutant enzymes contained only half of the cobalt complement of wild-type <i>Pt</i>NHase, but were able to hydrate acrylonitrile with <i>k</i><sub>cat</sub> values of 120 ± 6, 29 ± 3, and 14 ± 1 s<sup>−1</sup> for the αCys108His, Ser, and Met mutant enzymes, respectively. As all three of these mutant enzymes are catalytically competent, these data provide the first experimental evidence that transient disulfide bond formation is not catalytically essential for NHases.
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