Structural Biochemistry/Enzyme/Affinity Labels (reactive substrate analogs)

Definition
Affinity label or reactive substrate analog are molecules that have a similar structure to the substrate allowing them to covalently bind to the active site of the enzyme. They react with the substrate, inhibiting the enzyme. Essentially, affinity labels mimic the functional properties of the substrate, but consequently irreversibly inhibits the enzyme through the strong covalent bonding. Because of its similarity to the substrate, it initially binds at the active site; however, the covalent bonds it forms irreversibly modify the enzyme. They are much more specific than group specific inhibitors. They are usually highly reactive. Also, there will be alkylate nucleophilic amino acids in the enzyme. However, their properties are rather non-selective and toxic, making them not as useful in many situations.

Substrate analogs that bind to active sites of enzymes to form product of covalent bond that is stable on binding site to inhibit chemical reaction; irreversible enzyme inhibitor that react with functional group of enzyme and has more specificity for enzyme.

Use of Affinity Labels
Affinity labelling can be used to localize a given site within a protein of a known amino acid sequence. It allows comparisons to be made between the substrate sites of the enzyme in the solution and the sites localized within the crystalline form through X-ray diffraction. This can test whether the compound that is bound irreversibly at the substrate site can influence the enzyme conformation, subunit interaction, or the reactivity of other ligand sites.

Example of an Affinity Label
Tosyl phenylalanyl chloromethyl ketone is a substrate analog for chymotrypsin. This molecules binds at the active site and then react irreversibly with histidine residue at that site, inhibiting the enzyme.