Specificity is a property of the enzyme and describes how restrictive the enzyme is in its choice of substrate; a completely specific enzyme would have only one substrate.
The specificity of the serine proteases is usually not very high since they have similar active sites and act through the same proteolytic mechanism.
Consequently, a single serine protease may act on various substrates although at different rates. How the substrate fits the active site of the enzyme is of crucial importance to the outcome of the enzyme-substrate reaction. The bond to be cleaved must have a specific orientation relative to the amino acid side chains of the catalytic triad. The most important factor governing the fit of a substrate for an enzyme is the amino acid sequence around the bond to be cleaved.
Trypsin cleaves amides and esters of the basic amino acids arginine and lysine. Thrombin has a similar preference, but is more specific for arginine than for lysine.
Selectivity is a property of the substrate and indicates the degree to which the substrate is bound to and cleaved by different enzymes. The best measure for selectivity is given by the ratio kcat/Km. Synthetic substrates are considerably smaller than the natural substrates and can usually be cleaved by more than one enzyme, i. e. synthetic substrates are not completely selective. The explanation for this is that large substrates such as fibrinogen not only interact with the active site but also with exterior domains of the enzyme. Such interactions allow substrates to discriminate between different serine proteases and fibrinogen thus becomes highly selective for thrombin.
Selectivity Tables
The selectivity data of the table have been compiled to permit the investigator to understand how a contaminating enzyme would influence the enzyme-substrate reaction under study. Another way of expressing this is to say that the table shows the relative reactivities of two or more enzymes on one particular substrate. The table should be read horizontally. Each row represents the reactivity of a substrate designated for use with a particular enzyme, indicated to the left, relative to other relevant enzymes.
Example: The set of data in the top row shows the relative reactivity of the thrombin substrate S-2238 with various enzymes. All the experiments were performed using the same buffer, i.e. the one most appropriate for the reaction between thrombin and chromogenic substrate S-2238. In addition, the substrate concentration was always the same, or 2 x Km for the reaction of chromogenic substrate S-2238 with thrombin. The concentrations of the different enzymes are given in Table 2 and are related to the plasma concentration of the corresponding zymogen. The reactivity of chromogenic substrate S-2238 with thrombin, measured as the time-dependent increase in absorbance (ΔA/min), is given the value 100% (the actual value of ΔA/min is given in brackets). The reactivities of chromogenic substrate S-2238 with the enzymes FXa, FXIa, APC, plasmin, single chain t-PA, plasma kallikrein, and C1s have then been related to the reactivity of chromogenic substrate S-2238 with thrombin, and proved to be 5, 5, 40, 5, 5, 60, and 2%, respectively.