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Identification Of An Active Site-Bound Nitrile Hydratase Intermediate Through Single Turnover Stopped-Flow Spectroscopy, Natalie Gumataotao, Misty L. Kuhn, Natalia Hajnas, Richard C. Holz

Richard C. Holz

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 …