Rapid mixing transient kinetic methods

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. 

The kinetic constants k+ and k-1, which are properties of the molecules A and B and their interaction under specific conditions, are more difficult to determine experimentally than their ratio, which does not require resolving rapid transients to measure. One method to measure rate constants is by stopped-flow experiments, in which small reacting volumes are rapidly mixed and reaction progress followed, usually using some spectrophotometric assay. 

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 two prominent transient-kinetic mixing methods are stopped flow and rapid chemical quench flow. In the stopped flow, the... 

The kinetics of the transient phase of the hydrolysis of maltodextrin (average d.p. 11) by R. niveus glucoamylase have been studied using a fluorescent stopped-flow method. The fluorescence decreased rapidly on mixing solutions of the enzyme and the substrate, but slowly reappeared as the reaction proceeded the two phases are considered to represent the formation of an enzyme-substrate complex and the release of the free enzyme on breakdown of the complex into products. The importance of tryptophanyl residues at the subsite of R. niveus glucoamylase has been studied by modifying them with A-bromosuccinimide. ... 

The most useful approaches for obtaining information regarding the existence of intermediates and their lifetimes are fast reaction methods that mix enzyme and substrate within milliseconds, which permits the observation of single turnover events by various spectroscopic methods. Alternatively the reaction is rapidly quenched at known time intervals and its progress is analyzed chromatographically. In many cases in which an intermediate accumulates to the level of the enzyme concentration, such methods reveal the presence of burst kinetic that feature the rapid buildup of the intermediate in the transient phase followed by its slower rate of formation/decay in the steady state. The simplest kinetic scheme consistent with this phenomenon is given by... 

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