Kinetic studies with alternative other enzymes

Comparisons of the kinetic coefficients in Eq. (1) obtained from initial rate measurements with alternative substrates have given a considerable amount of information about reaction pathways as well as indications of the molecular basis of specificity (60). This approach, much used for proteolytic enzymes, has been exploited particularly with the alcohol dehydrogenases, which catalyze the oxidation of a variety of primary and secondary alcohols (61). While several other dehydrogenases have been studied in this way, most of the results have been reported only as apparent maximum rates and apparent Km values for the alternative substrate, which restricts the amount of information that can be derived. 

However, many enzymes show higher activity in mixtures of organic solvents with water than in pure water [23] and further, the native cellular microenvironment of enzymes is typically composed of lipids, proteins and other substances in addition to water. Since, in aqueous systems, the rate-determining step is often substrate diffusion in the vicinity of the active site of the enzyme [24, 25], supercritical fluid solvents have been perceived as an advantageous alternative. Kinetic studies showed that diffusion is still rate limiting, but that supercritical-fluid diffusivities were beneficial to the reaction rate [26-29]. 

There are three basic methods for carrying out alternative substrate inhibition studies. In the first, the investigator seeks to observe numerical changes in the coefficients of the double-reciprocal form of the enzyme rate expression in the presence and absence of the alternative substrate. For some mechanisms, only certain coefficients will be altered. This method requires extremely accurate estimates of the magnitudes of the coefficients and should always be supplemented with other kinetic probes . 

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