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A Novel Inhibitor for Fe-type Nitrile Hydratase: 2-Cyano-2-propyl Hydroperoxide

2003; American Chemical Society; Volume: 125; Issue: 38 Linguagem: Inglês

10.1021/ja035018z

1943-2984

Masanari Tsujimura, Masafumi Odaka, Hiroshi Nakayama, Naoshi Dohmae, Hiroyuki Koshino, Tadao Asami, Mikio Hoshino, Koji Takio, Shigeo Yoshida, Mizuo Maeda, Isao Endo,

Microbial metabolism and enzyme function

Nitrile hydratase (NHase) is a non-heme iron or non-corrin cobalt enzyme having two post-translationally modified ligand residues, cysteine-sulfinic acid (αCys112-SO2H) and -sulfenic acid (αCys114-SOH). We studied the interaction between Fe-type NHase and isobutyronitrile (iso-BN) which had been reported as a competitive inhibitor with a Ki value of 5 μM. From detailed kinetic studies of the inhibitory effect of iso-BN on Fe-type NHase, we found that authentic iso-BN was hydrated normally and that the impurity present in commercially available iso-BN inhibited NHase activity strongly. The inhibitory compound induced significant changes in the UV−vis absorption spectrum of NHase, suggesting its interaction with the iron center. This compound was purified by using reversed-phase HPLC and identified as 2-cyano-2-propyl hydroperoxide (Cpx) by 1H and PFG-HMBC NMR spectroscopy. Upon addition of a stoichiometric amount of Cpx, NHase was irreversibly inactivated, probably by the oxidation of αCys114-SOH to Cys-SO2H. This result suggests that the −SOH structure of αCys114 is essential for the catalytic activity. The oxygen atom in Cys-SO2H is confirmed to come from the solvent H2O. The oxidized NHase was found to induce the UV−vis absorption spectral changes by addition of Cpx, suggesting that Cpx strongly interacted with iron(III) in the oxidized NHase to form a stable complex. Thus, Cpx functions as a novel irreversible inhibitor for NHase.