In any enzyme-catalyzed reaction, the first step is the formation of an enzyme-substrate complex in which the substrate or substrates bind to the active site, usually noncovalently. Specificity of binding comes from the close fit of the substrate within the active-site pocket, which is due primarily to van der Waals interactions between the substrate and nonpolar groups on the enzyme, combined with complementary arrangements of polar and charged groups around the bound molecule. This fit is often so specific that even a small change in the chemical composition of the substrate will abolish binding. Enzyme-substrate dissociation constants range from about 10^-3 M to 10^-9 M; the lower the value the more tightly the substrate is bound. It is important that an enzyme does not hold onto its substrates or products too tightly because that would reduce its efficiency as a catalyst: the product must dissociate to allow the enzyme to bind to another substrate molecule for a new catalytic cycle.

Formation of a specific complex between the catalyst and its substrates does more than just account for the specificity of most enzymatic transformations: it also increases the probability of productive collisions between two reacting molecules. All chemical reactions face the same problem: the reacting molecules must collide in the correct orientation so that the requisite atomic orbitals can overlap to allow the appropriate bonds to be formed and broken. If we consider, in general terms, that a molecule can have a "reactive side" where the chemical changes take place and an "unreactive side" where they do not (at least immediately; the molecule may rearrange during the reaction), then in a simple reaction in which two molecules combine, both of them must collide reactive side-to-reactive side. Any other orientation and the collision will be non-productive. Thus, if both molecules first bind to an enzyme active site, and do so in such a way that their reactive portions are juxtaposed, the probability of a reaction is optimized. In solution, when two molecules collide but do not react they bounce off each other more or less randomly. On the enzyme, however, once the first reactive molecule has bound, it will stay there for some time, waiting for the second to come along. If that molecule does not bind productively, the first one may still remain associated with the enzyme (depending on the affinity constant) long enough for many other collisions to be tried.

In addition, enzyme active sites may even have evolved to attract their substrates so that finding the active site is not a random process. Most biological molecules are charged (so they can be retained within a cell by their insolubility in the hydrophobic membrane that surrounds it). Although active sites have exposed hydrophobic patches, the overall electrostatic field produced by the protein with all its polar and charged groups can yield an electrostatic potential with a net charge in the active-site region (Figure 2-22). It is possible that this net potential may "draw" the substrate into the oppositely charged active site, increasing the probability of productive binding (Figure 2-23a). Other ways in which electrostatics can aid in binding are illustrated in Figure 2-23b and 2-23c.

In most cases, there is a further critical factor in the facilitation of reactions by enzyme active sites. Usually, the enzyme itself supplies one or more of the chemical groups that participate in catalysis. Groups that are already part of the active-site structure in the folded protein before the substrate binds are already oriented properly for catalysis, or become so as the enzyme binds its substrate. The folding energy of the protein has already paid most, if not all, of the cost of positioning these groups, so there will be no unproductive collisions because they are in the wrong orientation. Every substrate molecule that forms the enzyme-substrate complex will therefore be exposed to an environment in which the catalytic groups are positioned correctly, relative to the substrate, for the desired reaction to take place.

In fact, the reactive portion of the substrate need not be the part that is used to hold it at the enzyme surface. An enzyme can recognize and interact with the remote parts of the substrate molecule, parts not involved in the chemistry, and use these interactions to hold and orient the substrate. This is a general principle: enzyme active sites consist of a specificity sub-site and a reaction sub-site, and in these protein groups are positioned around different parts of the substrate. In the specificity sub-site the enzyme uses polar and nonpolar groups to make weak interactions with the substrate; in the reaction sub-site other groups on the enzyme carry out the chemistry (Figure 2-24). In some cases the same amino-acid residue may participate in both specific substrate binding and catalysis. This design feature makes excellent sense. During the catalytic reaction, portions of the substrate molecule will undergo changes in geometry, charge and covalent bonding. If an enzyme had binding interactions with parts of the substrate that had to undergo rearrangement, those interactions would have to be broken before the substrate could change its structure, which could slow the enzyme down. The dual nature of enzyme active sites is being exploited in medicine and industry to design new catalysts. Amino-acid changes can often be made in the specificity sub-site of an enzyme without affecting its catalytic sub-site. So an enzyme that originally catalyzed a reaction involving positively charged substrates, for example, can sometimes be altered to perform exactly the same chemistry on new substrates that are negatively charged.

gated binding: binding that is controlled by the opening and closing of a physical obstacle to substrate or inhibitor access in the protein.

reaction sub-site: that part of the active site where chemistry occurs.

specificity sub-site: that part of the active site where recognition of the ligand takes place.

Figure 2-22 The electrostatic potential around the enzyme Cu,Zn-superoxide dismutase. Kindly provided by Barry Honig and Emil Alexov.