Transient-state kinetic analysis

Transient-state kinetic analysis is most commoifly based upon stopped-flow methods where an optical signal is used to follow the time dependence of a reaction however, it is often difficult or impossible to rigorously interpret the optical signal. For example, if the absolute extinction coefficients and concentrations of species contributing to the optical signal are not known, then the reaction pathway cannot be determined unambiguously. Some fast reactions do not result... 

Johnson KA. Transient-state kinetic analysis of enzyme reaction pathways. The Enzymes. 1992 XX 1-61. 

Transient-State Kinetic Analysis of Enzyme Reaction Pathways... 

The use of pH variation and isotope effects in transient kinetics can be illustrated with a recent study on dihydrofolate reductase. Analysis by steady-state methods had indicated an apparent p/fa of 8.5 that was assigned to an active site aspartate residue required to stabilize the protonated state of the substrate (59). In addition, it was shown that there was an isotope effect on substitution of NADPD (the deuterated analog) for NADPH at high pH but not at low pH, below the apparent p/fa This somewhat puzzling finding was explained by transient-state kinetic analysis. Hydride transfer, the chemical reaction converting enzyme-bound NADPH and dihydrofolate to NAD+ and tetrahydrofolate, was shown to occur at a rate of approximately 1000 sec at low pH. The rate of reaction decreased with increasing pH with a of 6.5, a value more in line with expectations for an active site aspartate residue. As shown in Fig. 14, there was a threefold reduction in the rate of the chemical reaction with NADPD relative to NADPH. Thus direct measurement of the chemical reaction revealed the full isotope effect. 

The application of transient kinetic methods to the solution of enzyme mechanisms has increased dramatically due to recent advances in instrumentation and in the overexpression and purification of new enzymes. Transient kinetics are becoming the method of choice for evaluation of site-directed enzyme mutants and for detailed questions regarding the relationships between protein structure and observable function. In conjunction with advances in methods of structural and genetic analyses, transient-state kinetic analysis forms the basis for what might be called the new enzymology. ... 

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