Transient kinetics, enzyme reactions rapid reaction techniques

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). 

Valuable insights into how DNA polymerases process their substrates were obtained as a result of detailed kinetic studies of the enzymes. Benkovic and coworkers employed rapid quenching techniques to study the kinetics of transient intermediates in the reaction pathway of DNA polymerases [5]. Intensive studies revealed that E. coli DNA polymerase I follows an ordered sequential reaction pathway when promoting DNA synthesis. Important aspects of these results for DNA polymerase fidelity are conformational changes before and after the chemical step and the occurrence of different rate-limiting steps for insertion of canonical and non-canonical nucleotides. E. coli DNA polymerase I discriminates between canonical and non-canonical nucleotide insertion by formation of the chemical bond. Bond formation proceeds at a rate more than several thousand times slower when an incorrect dNTP is processed compared with canonical nucleotide insertion. 

Transient-kinetic techniques most often rely on the rapid mixing of reactants with enzyme to initiate the reaction. This mixing is essential so that all enzyme molecules start reaction in synchrony with one another therefore, the time dependence of the observable reactions dehnes the kinetics of interconversion of enzyme intermediate states. Because mixing requires a hnite amount of time, conventional methods are limited in their ability to measure very fast reactions. For example, a typical value for the dead time of a stopped-flow instrument is approximately 1 ms, which is because of the time it takes to hll the observation cell. Thus, reactions with a half-life of less than 1 ms (rate > 700 s ) are difficult to observe depending on the signal to noise... 

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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