Transient kinetics, enzyme reactions time course

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)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... 

Chance (1943) and Theorell Chance (1951) observed the formation and decomposition of complexes of enzymes with substrates and products by following changes in light absorption. In these pioneering studies the theories and techniques of pre-steady-state kinetics were only applied to reactions in which the complexes had distinct absorption spectra. It became apparent that transients of a much wider range of enzyme reactions could be studied when the initial rate of product formation is analysed (Gut-freund, 1955). Observations with a time resolution of milliseconds showed that there are often three distinct phases in product formation. These are determined in turn by the rate of formation of the enzyme-substrate complex, the enzyme-product complex and of free product. Of course, as we shall see, the most fruitful investigations into enzyme mechanisms resulted when it was possible to combine the observation of transients of product formation with those of spectral changes of complexes. 

So far, examples to illustrate experimental methods for following the time course of the approach to steady states and of their kinetic interpretation have been restricted to enzymes which do not have a natural chromophore attached to the protein although reference has been made to the classic studies of Chance with peroxidase (see p. 142). Qearly the application of these techniques to the study of enzymes with built in chromophores, such as the prosthetic groups riboflavine, pyridoxal phosphate or haem, contributed considerably to the elucidation of reaction mechanisms. However, the progress in the identification of the number and character of intermediates depended more on the improvements of spectral resolution of stopped-flow equipment than on any kinetic principles additional to those enunciated above. This is illustrated, for instance, by the progress made between the first transient kinetic study of the flavoprotein xanthine oxidase by Gutfreund Sturtevant (1959) and the much more detailed spectral analysis of intermediates by Olson et al. (1974) and Porras, Olson Palmer (1981). 

The steady-state and rapid equilibrium kinetics do not give detailed information on the existence of multiple intermediates or on their lifetimes. Such information is provided by fast (or transient) kinetics. The methods can be divided in two categories rapid-mixing techniques (stopped-flow, rapid-scanning stopped-flow, quenched flow) which operate in a millisecond time scale and relaxation techniques (temperature jump, pressure jump) which monitor a transient reaction in a microsecond time scale. Most of the transient kinetic methods rely on spectrophotomet-rically observable substrate changes during the course of enzyme catalysis. 

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