Transient kinetics, enzyme reactions reaction intermediates identification

Identification of radical 3 as a species that is present in the steady-state phase of the reaction does not prove that it is an intermediate—it could be a species that is peripheral to the real reaction mechanism. Proof that a species is an intermediate requires a demonstration that it is kinetically competent to participate in the mechanism. In the case of a metastable radical, the usual procedure is to conduct transient kinetic studies using a rapid mixing apparatus equipped to quench samples by spraying them into liquid isopentane. The frozen aqueous samples (snows) from the timed cold quenches are then packed into EPR tubes and analyzed spectroscopically. Simple mixing of enzyme with SAM and lysine followed by freeze-quenching on the millisecond time scale does not work because the activation by SAM takes about 5 s. However, a preliminary mix of enzyme with SAM and [2- C]lysine, aging of the solution for 5 s within the apparatus. 

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 concept of ordered interactions of substrates with the enzyme and ordered dissociation of the products was advanced by Koshland in 1954. From then through the 1960s the introduction of stopped-flow techniques and relaxation methods allowed rapid reactions to be followed and the identification of transient intermediates, from which much more complex kinetic analyses have emerged (Fersht,1977). 

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