Enzyme Dynamics in Nonaqueous Media at Subzero Temperatures
Despite the continuous development of new data acquisition techniques that reduce the time and improve the precision of recording, it is not possible to directly observe the nature and structure of most intermediates or to determine rates of conformational changes that accompany each activation process, stepping the reaction pathway either in solution or in the crystalline state. In order to circumvent this difficulty, we set up a procedure to investigate enzyme-catalyzed reactions at subzero temperatures in which the reaction rates are dramatically slowed according to the classic Arrhenius relation. In particular, reactions that normally occurred in a few seconds or minutes could be sufficiently slowed to permit enzyme—substrate intermediates to be stabilized and eventually isolated by column chromatography, and then studied by available spectroscopic techniques, including x-ray diffraction by enzyme crystals. The physicochemical basis of the procedure as well as its preliminary results have been reviewed elsewhere (1–3).
KeywordsElementary Step Subzero Temperature Nonaqueous Medium Enzyme Dynamics Collision Complex
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