An electron is in its ground state meaning it is in its lowest energy state, in other words, an electron is in its excited state whenever it is not in its ground state. An excited state of a molecule is known to have higher energy levels than its ground state. The third law of thermodynamics states that the system is at its ground state when it is at absolute zero degree in temperature, which causes the entropy of the reaction to be determined by the degeneracy of that ground state. However, some systems will have zero entropy due to its physical/chemical properties.
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 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 below, 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. File:Pre-steady-state.pdf
The steady state assumption was proposed by George Briggs and John Haldane in 1942. In this assumption, the concentrations of the intermediates of a reaction remain the same even when the concentrations of starting materials and products are changing. Steady state occurs when the rate of formation and breakdown of the intermediate are equal. The steady state assumption relies on the fact that both the formation of the intermediate from reactants and the formation of products from the intermediate have rates much higher than their corresponding reverse reactions. In other words, steady state assumes that k1>>k-1 and k2>>k-2.