Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy
Identification of an Active Site-bound Nitrile Hydratase Intermediate through Single Turnover Stopped-flow Spectroscopy
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DOI：
10.1074/jbc.m112.398909
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发表时间：
2013-05-31
影响因子：
4.8
通讯作者：
Holz, Richard C.
中科院分区：
文献类型：
--
作者：
Gumataotao, Natalie;Kuhn, Misty L.;Holz, Richard C.
以甲基丙烯腈为底物，研究了马红球菌TG328-2(ReNHase)铁型水合腈酶(ReNHase)的停流动力学数据。多次周转实验提出了一个三步动力学模型，该模型允许底物的可逆结合、中间体的存在和产物的形成。由这些数据测得的微观速率常数与稳态数据吻合较好，证实了停流法适用于该反应。采用单周转停流实验对催化中间体进行鉴定。这些数据完全符合三步动力学模型。在反应的0.005-0.5 S之间获得的独立吸收光谱显示，随着Fe3+的亚色移位，在375,460和550 nm处的吸光度显著增加
Stopped-flow kinetic data were obtained for the iron-type nitrile hydratase from Rhodococcus equi TG328-2 (ReNHase) using methacrylonitrile as the substrate. Multiple turnover experiments suggest a three-step kinetic model that allows for the reversible binding of substrate, the presence of an intermediate, and the formation of product. Microscopic rate constants determined from these data are in good agreement with steady state data confirming that the stopped-flow method used was appropriate for the reaction. Single turnover stopped-flow experiments were used to identify catalytic intermediates. These data were globally fit confirming a three-step kinetic model. Independent absorption spectra acquired between 0.005 and 0.5 s of the reaction reveal a significant increase in absorbance at 375, 460, and 550 nm along with the hypsochromic shift of an Fe3+