The study of enzyme mechanisms by a combination of cosolvent, low-temperature and high-pressure techniques

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Abstract
It is generally assumed that the mechanism of enzyme-catalysed reactions would be defined if all the intermediates, complexes and conformational states of each enzyme could be characterized, and the rate-constants for their inter-conversion recorded. In spite of the introduction during the last decades of methods for rapid data acquisition, which permit detection of the number and sequence of intermediates and complexes, measurement of rate-constants, identification of the types of catalysis involved, etc., at best a semi-quantitative understanding of the mechanism of enzyme-catalysis is obtained. The definition of the exact chemical nature of intermediates and complexes is missing because techniques establishing the structures are restricted to the study of transient states which are stable for periods that exceed the half-life of most of typical intermediates. In such conditions, while conformational changes are obviously an essential feature of enzyme activity, the conformational basis of such activity cannot be understood at the molecular level, and enzyme catalysis is still termed a ‘miracle’ compared to the rate-enhancements and specificity of ordinary chemical catalysts.