Active Site
 
The folding of a polypeptide chain that produces the final protein structure of an enzyme also leads to formation of the active site. From X-ray crystallography studies, it is apparent that the active site of an enzyme is a groove, cleft, or pocket that has access to the solvent and forms only a small part of the total solvent-accessible surface of the protein. The relatively large sizes of enzymes are undoubtedly due to the need to obtain, at the active site, the correct spatial relationships of the amino acid residues that are involved in binding of substrates, catalysis, and the release of products, as well as for any conformational changes associated with these steps. The binding of substrates or inhibitors at the active site pocket of an enzyme involves matching up of the nonpolar groups of the substrate with the nonpolar side-chains of amino acid residues, hydrogen bonding between the polar appropriate groups on the substrate with the backbone NH and CO groups within the active site, and even salt bridge formation. For substrates, these initial interactions are followed by the conformational changes that lead to the formation of the transition-state complex (see Transition State Analogue) and the chemistry for catalyzing the reaction brought about by reactive groups with the correct alignments. These may be the acidic, basic, and nucleophilic groups of the protein component of the enzyme, or the electrophilic groups of a prosthetic group (see Coenzyme, Cofactor).
Active Site-Directed Irreversible Inhibitors
Active site-directed irreversible inhibitors of enzymes are also known as active site-direcetd inactivating reagents, affinity labels, and photoaffinity labels. They combine the features of a substrate, or substrate analogue, with those of a group-specific reagent, as in affinity labeling, and have been used to determine the amino acid residues that are present in the active site and involved in enzymic catalysis. They are capable of binding specifically and reversibly at the active site of an enzyme and then causing inactivation through time-dependent covalent modification of an adjacent amino acid residue. The functional group of the inhibitor is usually an electrophile that can interact with an appropriately positioned nucleophile of the enzyme, to generate a covalent bond between them. The electrophilic groups include epoxides and a-haloketones. Since these compounds are reactive in solution, they could also cause some nonspecific enzyme modifications.
The action of an active-site directed irreversible inhibitor I can be illustrated by
E+I ⇔ EI  ⇒E-I             (1)
 where EI represents a Michaelis complex (see Michaelis–Menten Kinetics) and E – I denotes the covalently cross-linked form of the complex. On the basis of this formulation, it would be expected that at the early stages of the interaction, I would behave as a competitive inhibitor with respect to the substrate. Examples of the action of an active-site directed irreversible inhibitors are the acetylation of amino acid residues at the active site of prostaglandin synthase by aspirin (acetyl salicylate) (1) and the alkylation by L-TPCK (N-tosylphenylalanine chloromethyl ketone) of a histidine residue at the active site of a-chymotrypsin (2).
Photoaffinity labels, such as diazoketones and aryl azides, introduce a greater degree of specificity to the modification of amino acid residues, as they are not reactive in solution. It is only after the reversible interaction of the affinity label at the active site of an enzyme, and exposure of the resulting complex to light of the correct wavelength, that a highly reactive group is formed. Such treatment with diazoketones and aryl azides leads to the formation of carbenes and azines that are extremely reactive and can add across O H bonds of unionized carboxyl groups or unsaturated carbon–hydrogen bonds (3).
References
1. G. J. Roth, N. Stanford, J. W. Jacobs, and P. W. Majerus (1977) Biochemistry 16, 4244–4248.  
2. C. Walsh (1979) Enzymatic Reaction Mechanisms, W. H. Freeman and Company, San Francisco,  Calif., p. 86. 
3. V. Chowdry and F. H. Westheimer (1979) Ann. Rev. Biochem. 48, 293–325.