Pre-steady-state Kinetics
In the first moment after an enzyme is mixed with substrate, no product has been formed and no
intermediates exist. The study of the next few milliseconds of the reaction is called pre-steady-state
kinetics. Pre-steady-state kinetics is therefore concerned with the formation and consumption of
enzyme–substrate intermediates (such as ES or E*) until their steady-state concentrations are reached.
This approach was first applied to the hydrolysis In the figure to the left, the enzyme produces E*
reaction catalysed by chymotrypsin. Often, the rapidly in the first few seconds of the reaction.
detection of an intermediate is a vital piece of The rate then slows as steady state is reached.
evidence in investigations of what mechanism an This rapid burst phase of the reaction measures
enzyme follows. For example, in the ping–pong a single turnover of the enzyme. Consequently,
mechanisms that are shown above, rapid kinetic the amount of product released in this burst,
measurements can follow the release of product P and shown as the intercept on the y-axis of the
measure the formation of the modified enzyme graph, also gives the amount of functional
intermediate E*. In the case of chymotrypsin, this enzyme which is present in the assay.
intermediate is formed by an attack on the substrate
by the nucleophilic serine in the active site and the
formation of the acyl-enzyme intermediate.
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