Specificity is one of the main characteristics of enzymes. It prevents the formation of by-products which happens with chemical catalysts. Specificity may be observed on the one hand in the type of reaction catalyzed by the enzyme, and on the other hand in the substrate of the reaction.
Reaction-Related Specificity:
A given substrate, an α-amino acid for example, can undergo various transformations. Each of the 3 reactions of figure 2-23 is catalyzed by a different enzyme although the substrate is the same.
Substrate-Related Specificity:
Specificity may be very broad (the enzyme acts on a wide variety of compounds) or on the contrary, it may be very narrow (the enzyme acts on one substrate only); there are also numerous intermediate possibilities (the enzyme acts on a family of compounds). We will take the example of maltose which, as indicated by its name, hydrolyzes maltose (structure shown in figure 4-15) with formation of two molecules of glucose.
Maltase is a hydrolase, catalyzing a reaction of the type A — B + H2O → A — OH + B — H. But a large number of linkages can be hydrolyzed (ester linkages, peptide linkages, oside linkages etc.) and there is one type of specific enzyme for each type of linkage (esterases, peptidases, osidases etc.). Maltase is an osidase because it breaks the bond joining two monosaccharides.
These two monosaccharides are in fact two glucose molecules; maltase is therefore a glucosidase. They are coupled by a 1-4 linkage which precisely, maltase can hydrolyze; it is therefore a 1-4 glucosidase. The linkage is of α-type, and glucose is of the D series, maltase is therefore a α, D, 1-4 glucosidase.
1. Specificity Related to the Nature of the Linkage:
As just mentioned in the case of hydrolases, there are osidases, esterases, amidases etc. according to the type of the linkage.
2. Group Specificity:
Among esterases for example, one may distinguish carboxy-esterases — which are specific of ester linkages where organic acids are involved — from phospho-esterases which hydrolyze phosphoric esters.
As shown by figure 2-24, the latter are again sub-divided in phosphomonoesterases or phosphatases which split the phosphomonoester linkages (notably in the metabolism of carbohydrates), and phosphodiesterases which hydrolyze one of the ester linkages of a diesterified phosphoric acid (example: ribonucleases and deoxyribo-nucleases; see figures 6-10 to 6-12).
Among proteolytic enzymes, or proteases, which hydrolize the peptide linkages; there are exopeptidases which act at the end of chains and detach the N — or C-terminal amino acid, and endopeptidases which hydrolyze the peptide linkages within the chains.
As shown in figure 2-25, some endopeptidases have a specificity: pepsin preferentially splits the peptide linkages where an aromatic amino acid (Phe, Tyr) is involved by its amino group; trypsin preferentially hydrolyzes the peptide linkages where a basic amino acid like arginine or lysine is involved by its carboxylic group, and chymotrypsin breaks with greater ease the linkages where an aromatic amino acid is involved by its carboxyl.
3. Absolute Specificity for One Substrate Only:
Some enzymes act on only one substrate: urease acts only on urea; arginase only on L-arginine; fructose 1-6-bisphosphatase only on fructose 1-6 bisphos-phate; carbonic anhydrase only on CO2 etc.
Some oxidoreductases and transferases manifest even a narrower specificity concerning not only the substrate or the group of substrates they can recognize, but also the acceptor of the group whose transfer they catalyze. For example, 3-phosphoglyceraldehyde dehydrogenase (see fig. 4-27) catalyzes the transfer of hydrogen only from 3-phosphoglyceraldehyde (substrate) to NAD+ (acceptor).
4. Stereo Specificity:
Several enzymes can distinguish between two optical isomers and act only on one of them. Thus, a L-amino acid oxidase (see fig. 7-2) recognizes only a L-amino acid and the transformation of a D-amino acid requires a D-amino acid oxidase.
Proteolytic enzymes like trypsin or chymotrypsin will catalyze the hydrolysis or their substrates only if they are formed of L-type amino acids and have no activity on substrates derived from D-type amino acids.
Some enzymes can distinguish between the α- and β-conformations in osidc linkages; there are, for example, α- and β-glucosidases, α- and β-galactosidases, etc.
Other enzymes have a stereospecificity vis-a-vis cis-trans isomers. Thus, fumarase allows the addition of one molecule of water on the double bond of fumaric acid (trans), but not on that of maleic acid (cis), (see fig. 2-26).
A stereospecificity which is even more subtle is observed vis-a- vis the substances of the general formula Cx1x2yz, like glycerol or citric acid (see fig. 2-27). From the organic chemistry point of view such substances are symmetric (they have a plane of symmetry) but they behave like asymmetric substances as substrates of certain enzymatic reactions (in other words x1 for example, can be attacked in preference to x2).
In fact the free substrate is symmetric, but it becomes asymmetric during the formation of the E — S complex and when 3 (at least) functional groups of the substrate must interact with 3 groups of the active site of the enzyme. If the 3 groups of the substrate are all different, the substrate can bind to the enzyme in one way only (model I), and it is clear that x1 and x2 are no longer equivalent because one of them is bound and the other not.
If on the contrary, x1 and x2 are among the 3 functional groups of the substrate involved in the formation of the complex with the enzyme, the asymmetry is less evident but not any the less real (model II); to make sure, one may consider that the left hand represents the enzyme, with the thumb (Z), the index (X1) and the middle finger (X2) at the active site, and that the right hand represents the substrate with the 3 same fingers corresponding respectively to z,x1 and x2; this shows that only one orientation is possible; therefore, x1 and x2, by the fact that they are bound to groups of the enzyme which are not symmetric, cease to be equivalent, and one can understand that only one of them undergoes the transformation catalyzed by the enzyme.
This notion of stereo specificity has found a direct application called affinity labeling for the study of active sites of enzymes. The method consists in forming a complex between the enzyme and a steric analogue of the substrate on which a reactive chemical group has been grafted.
The analogue behaves as a competitive inhibitor and therefore forms with the enzyme, in a first step, a non- covalent complex. In a second step, the chemical group of the analogue reacts with an amino acid of the active site and the complex then transforms into a covalent complex. After proteolysis of the enzyme thus modified, one can identify the amino acid which has reacted with the analogue.
With this method it is therefore possible to locate the amino acids of the active site, involved either in the recognition of the substrate, or in the catalytic mechanism, and since it is based on this very important notion of stereo-specificity, it is applicable to all types of sites, particularly also to allosteric sites. Affinity chromatography is also a direct application of specificity.