Kinetics, the study of rates of chemical reactions, allows one to determine the detailed mechanistic steps of chemical reactions. One can determine affinities of substrates, products and inhibitors for an enzyme, catalytic mechanism, inhibition mechanism, role in coupled, sequential reactions, etc. In one of the simplest enzymatic mechanisms, an enzyme (E) and a substrate (S) combine to form an enzyme-substrate complex (ES), which decomposes to product (P) and enzyme.

One can simplify the rate equation by making several assumptions.Assumption 1. Assume that k-1>> k2. If this assumption is correct, the first step of the reaction is essentially in equilibrium. The second step is very slow compared to the first step.

Assumption 2. Assume steady state. The steady state assumption is that [ES] is constant. This assumption is true if the rate of formation of ES is the same as the rate of decomposition.

The resulting Michaelis-Menten equation is:

Vo = Vmax [S] / ( Km + [S] )

Or

1/vo = (Km/Vmax)1/[S] + 1/Vmax

Vo is the initial velocity of the reaction

Vmax = k2[Etot], is the maximal velocity of the reaction

[S] = substrate concentration

Km = (k-1 + k2 ) / k1. Km is known as the Michaelis Constant. Km is the substrate concentration at which the reaction velocity is half of the maximal velocity.

k2 is also know as kcat, the 'turnover number', which is the number is reaction processes per unit time, per enzyme.

The goal here is to determine kinetic parameters (Km and kcat) for a reaction catalyzed by a protease.

The mechanism and class of a serine protease are essentially independent of the specificity.

For example both a-chymotrypsin and trypsin are serine proteases, with nucleophilic serine residues and conserved catalytic triads.

Yet the S1 specificity of a-chymotrypsin differs from that of trypsin; a-chymotrypsin has S1 specificity for bulky aromatic residues (phenylalanine) while trypsin has S1 specificity for basic residues (arginine and lysine).