Tsujimura Masanari, Odaka Masafumi, Nakayama Hiroshi, Dohmae Naoshi, Koshino Hiroyuki, Asami Tadao, Hoshino Mikio, Takio Koji, Yoshida Shigeo, Maeda Mizuo, Endo Isao
Biomolecular Characterization Division, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
J Am Chem Soc. 2003 Sep 24;125(38):11532-8. doi: 10.1021/ja035018z.
Nitrile hydratase (NHase) is a non-heme iron or non-corrin cobalt enzyme having two post-translationally modified ligand residues, cysteine-sulfinic acid (alphaCys112-SO(2)H) and -sulfenic acid (alphaCys114-SOH). We studied the interaction between Fe-type NHase and isobutyronitrile (iso-BN) which had been reported as a competitive inhibitor with a K(i) value of 5 microM. 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 (1)H and PFG-HMBC NMR spectroscopy. Upon addition of a stoichiometric amount of Cpx, NHase was irreversibly inactivated, probably by the oxidation of alphaCys114-SOH to Cys-SO(2)H. This result suggests that the -SOH structure of alphaCys114 is essential for the catalytic activity. The oxygen atom in Cys-SO(2)H is confirmed to come from the solvent H(2)O. 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.
腈水合酶（NHase）是一种不含血红素的铁或不含咕啉的钴酶，具有两个翻译后修饰的配体残基，即半胱氨酸亚磺酸（αCys112-SO₂H）和亚磺酸（αCys114-SOH）。我们研究了铁型NHase与异丁腈（iso-BN）之间的相互作用，异丁腈已被报道为一种竞争性抑制剂，其抑制常数（Kᵢ）值为5微摩尔。通过对异丁腈对铁型NHase抑制作用的详细动力学研究，我们发现纯的异丁腈能正常水合，而市售异丁腈中存在的杂质会强烈抑制NHase活性。这种抑制性化合物使NHase的紫外可见吸收光谱发生了显著变化，表明它与铁中心发生了相互作用。该化合物通过反相高效液相色谱法纯化，并通过¹H和PFG-HMBC核磁共振光谱鉴定为2-氰基-2-丙基氢过氧化物（Cpx）。加入化学计量的Cpx后，NHase不可逆失活，可能是由于αCys114-SOH被氧化为Cys-SO₂H。这一结果表明αCys114的-SOH结构对催化活性至关重要。已证实Cys-SO₂H中的氧原子来自溶剂H₂O。发现氧化型NHase加入Cpx后会引起紫外可见吸收光谱变化，表明Cpx与氧化型NHase中的铁（III）强烈相互作用形成稳定复合物。因此，Cpx可作为NHase的一种新型不可逆抑制剂。
