In this article we will discuss about the mechanisms of enzymatic reactions.
While studying the kinetics of enzymatic reactions we noted that Michaelis was led to postulate the formation of an intermediate enzyme-substrate complex right at the beginning of this century. What was then only a hypothesis was later largely confirmed and proved.
The great disproportion between the size of the enzyme and that of the substrate obviously suggested that only a limited part of the enzyme participates in this interaction; this part is called active site (or active centre), or catalytic site. This is where the substrate binds and undergoes the transformation into reaction product (or products).
I. Active Site of Enzymes:
It will be seen later, that the formation of the enzyme-substrate complex is characterized by a specificity, and even a rather narrow stereo-specificity, due to the fact that the substrate molecule must have several functional groups in a suitable spatial configuration which will allow these groups to react with the corresponding functional groups of the enzyme.
These enzyme groups are not near one another from the point of view of the primary structure (sequence of amino acids) but they are so from the point of view of the three-dimensional conformation: the folding of the chain brings them close together to form the active site (or active centre); this explains why catalytic activity requires a native conformation and disappears upon denaturation of the protein.
The bonds involved in the formation of the enzyme-substrate complex are the same as those responsible for the spatial structure of proteins. They enable the union of certain groups of the substrate with certain groups (in other words, certain amino acids) of the enzyme.
But the notion of active site is not limited to the amino acids involved in these new bonds: for a better understanding, the amino acids of an enzyme molecule may be divided in 3 broad groups:
1. Amino acids involved in the first step of enzymatic catalysis, i.e. the amino acids which constitute the specificity site and ensure the recognition of the substrate to enable the reaction E + S 
E – S;
2. Amino acids involved in the second step and which participate in the chemical transformation of the substrate according to the reaction E – S → E + P. They form the catalytic part of the active centre;
3. Other amino acids which are necessary for maintaining the adequate conformation of the active centre, maintaining the adequate conformation of the allosteric sites (which control the functioning of the active centre), and adequate positioning of the enzyme within the cell (for example, association of the enzyme with another enzyme to form a multi-enzyme complex, or association with a membrane).
The active centre comprises amino acids of the first two groups. The study of active sites is rather delicate; in most cases it requires the simultaneous use of several methods. The comparative study of the fine three-dimensional structure of the enzyme (with the help of X-rays) when free or involved in the complex, provided valuable information in the case of crystallizable enzymes (lysozyme, for example).
We have also already seen that the study of enzymatic action as a function of pH enables in certain cases, the determination of the pK of a group implicated in the interaction. But chemical labelling methods have provided most of the accurate information available on the nature of amino acids which are part of the active sites.
After binding the reagent to one (or several) residue(s), the protein is hydrolyzed; this yields the label-carrying amino acid in a peptide, which enables its characterization and the determination of its position in the polypeptide chain. Obviously, one must make sure that the label did bind an amino acid belonging to the active site; this may be verified when the binding of the reagent causes a stoichiometric inactivation of the enzyme or on the contrary, when the binding of the reagent is prevented by a “protective” compound which is known to bind to the active site of the enzyme.
Very often, the active site of an enzyme contains an amino acid residue, which if not unique, is at least highly reactive and will react preferentially with the label (for example the seryl residue of esterases).
In certain cases the label can be the substrate itself, provided the E — S complex is formed by sufficiently stable covalent bonds (for example, phosphoglucomutase, which catalyzes the isomerization of glucose-6-phosphate into glucose-1-phosphate, can be labelled by phosphate and phosphoryl-serine can then be isolated), or a quasi-substrate capable of forming a stable covalent bond with the enzyme, such as di-isopropylfiuorophosphate, which was studied in connection with inhibitors (see fig. 2-8) and which binds to the seryl residues belonging to the active sites of a large number of esterases and proteases.
The use of bi-functional reagents combining with 2 groups of the active site of the enzyme could confirm that these groups can be part of peptide sequences very remote from one another in the primary structure but sufficiently close together in the spatial conformation, to enable the same reagent molecule to combine with both.
Very recently, with the possibility of isolating the gene of a given enzyme it has become possible to prepare enzyme molecules modified at will, by modification and then expression of this gene.
One can thus replace an amino acid of the active site by another or delete a part of the molecule. Based on the study of molecules thus modified one can determine the role of each amino acid of the active site or the function of various regions of the molecule.
The conformation of the active centre is not rigid. It has a great flexibility. The association of the substrate with the active centre induces very regularly a change of conformation which results in an induced adjustment or induced fit of the structure of the active centre.
The side chains which participate in the functioning of the active centre, either at the specificity centre, or in the catalytic part of the active centre, change position during the attachment of the substrate to form the enzyme-substrate complex, but very often also, during the catalytic transformation of the complex to form the reaction products.
These position changes of amino acids which constitute the active centre are not necessarily very considerable; they can be only of the order of a fraction of an angstrom (1Å = 1/10 000 000 mm); however, the structural reorganizations taking place at the active centre upon the binding of the substrate may sometimes correspond to position changes of the side chains of the amino acids of the active centre, attaining 5 to 10Å (i.e. 0.5 to 1 nm).
It is quite obvious that the mutations which will lead to an enzyme structure, in which the conformational changes taking place in the active centre and which are essential for the expression of catalytic activity are blocked, will cause an inactivation of the enzyme.
II. Mechanisms of the Catalysis:
As an example, figure 2-22 represents the mechanism of action of chymotrypsin, as it is presently admitted. This is a proteolytic enzyme which hydrolyzes certain peptide bonds in two steps: first, acyIation and then deacylation with regeneration of the enzyme as it was at the beginning of the reaction.
The catalytic part of the active site consists of 3 amino acids which are very distant from one another in the sequence (they occupy respectively the positions 57, 201 and 195) but close together in the three-dimensional conformation.
According to the classification given above, histidine, serine and aspartic acid are part of the catalytic site of the enzyme. This enzyme has a specificity for aromatic amino acids. The active site consists of a groove of hydrophobic character containing particularly methionine 192.
Some enzymes — particularly those responsible for the digestion of proteins in our digestive tract (pepsin, trypsin, chymotrypsin) — are synthesized in a catalytically inactive form called proenzyme or zymogen (pepsinogen, trypsinogen, chymotrypsinogen).
Their conversion into active enzyme consists of the cleavage of one or several peptide bonds, reaction which is itself catalyzed by an enzyme: for example, the transformation inactive chymotrypsinogen → active chymotrypsin is catalyzed by trypsin which breaks the bond arg 15 — ile 16 in chymotrypsinogen.
The catalytic site is already present in chymotrypsinogen, but the specificity site does not exist; cleavage by trypsin initiates a reorganization of the spatial structure which reveals the specificity site.
In reactions where there are 2 different substrates, both must be bound to the enzyme in such a way that their interacting groups are near one another, and the active centre of the enzyme must have a binding site for each substrate.
While studying the general principles of catalysis, we have seen that the enzyme increases reaction velocity.
Although it is not possible to explain, for each enzyme, how this is achieved, it may be said that in most cases two factors play a capital role:
1. The bonds established between enzyme and substrate cause modifications of the electrons’ distribution between the atoms of the substrate and can thus increase the reactivity of some groups of the substrate;
2. The enzyme, in binding 2 substrates in such a way that their respective interacting functional groups are near one another, increases the probability of effective collisions needed for the reaction to take place between the 2 substrates.
III. Role of Co-Factors:
We have seen — for example in the case of NAD – that a coenzyme is in most cases, one of the substrates of the reaction. But it is bound to the enzyme more strongly than an ordinary substrate, before and after the reaction (hence its name).
Moreover, it is restored to the initial state by a second reaction, so that the same coenzyme molecule can react successively with a very large number of molecules of the substrate (provided it is brought back to the initial state each time), and this explains why reference is sometimes made to the cyclic role of coenzymes.
This regeneration of the coenzyme can take place while it remains bound to the same apoenzyme or after its detachment (in the latter case the coenzyme can bind again to a molecule of the same apoenzyme after regeneration).
Sometimes, metal ions can act in the same manner, i.e. they may be reduced in a first reaction, then reoxidized in a second reaction, as in the case of cytochromes for example (see fig. 3-6a).
But in other cases, they can, by formation of a complex, enable the binding of the substrate to the enzyme; for example, it is believed that in some enzymatic reactions, Mg2+ ions form bridges between the phosphate groups of the substrate and the enzyme molecules and that Co2+ or Zn2+ ions are necessary for the bond between the dipeptide glycylglycine and the dipeptidase etc.
