Enzyme Kinetics - Pre-steady-state Kinetics

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 also referred to as Burst 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 reaction catalysed by chymotrypsin. Often, the detection of an intermediate is a vital piece of evidence in investigations of what mechanism an enzyme follows. For example, in the ping–pong mechanisms that are shown above, rapid kinetic measurements can follow the release of product P and measure the formation of the modified enzyme intermediate E*. In the case of chymotrypsin, this 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.

In the figure to the right, the enzyme produces E* rapidly in the first few seconds of the reaction. The rate then slows as steady state is reached. This rapid burst phase of the reaction measures a single turnover of the enzyme. Consequently, the amount of product released in this burst, shown as the intercept on the y-axis of the graph, also gives the amount of functional enzyme which is present in the assay.