Kinetic identification of reaction intermediates

1 Kinetic identification of reaction intermediates Singk ami multiple turnover of enzyme reactions 

For the purpose of the present discussion the term transient kinetics is applied to the time course of a reaction from the moment when enzyme and substrate are mixed, t=0, until either a steady state or equilibrium is established. The difference between the kinetic problems discussed in section 3.3 and in the present section is, respectively, the presence of catalytic as distinct from catalytic concentrations of enzyme. Here we are concerned with the stoichiometry of enzyme states. Transient kinetic experiments with enzymes can be divided into two types. The first of these (multiple turnover) is carried out under the condition that the initial concentrations of substrate and enzyme are Cs(0) Ce(0) and c it) can, therefore, be regarded as constant throughout the course of the reaction until a steady state is attained. Alternatively, in a single turnover reaction, when Cs(0) Ce(0), the transient formation and decay of reaction intermediates is observed until the overall process is essentially complete. These two possibilities will be illustrated with specific examples. In connection with a discussion of the approach to the steady state, in section 3.3 it was emphasized that, at t = 0, the concentrations of the intermediates, enzyme-substrate and enzyme-product complexes, are zero and, therefore, the rate of product formation is also zero. Under the experimental conditions used for steady state rate measurements and for enzyme assays, the first few seconds after the initiation of a reaction are ignored. However, when the experimental techniques and interpretation discussed below are used, events during the first few milliseconds of a reaction can be analysed and provide important information. With suitable monitors it is possible to follow the formation and decay of enzyme complexes with substrates and 

Jencks s (1989) comments in his minireview on How does a calcium pump pump calcium are worth quoting extensively as a testimony to the transient kinetic study of enzymes  

The investigations of individual steps of the reaction catalysed by the calcium ATPase that have been carried out in many laboratories have clarified what this enzyme does and have provided a good deal of information on how it does it. In contrast, relatively little has been learned about the enzyme from investigations of 

These comments apply to all enzyme mechanisms to varying degrees and particularly also to a problem closely related to the calcium pump, that of the myosin ATPase coupled to muscle contraction. The methods used to study the latter enzyme are discussed in some detail below and are also the ones which helped to elucidate the steps involved in the coupled process of ATP hydrolysis and calcium translocation. 

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