Affinity labelling

Abstract

Abstract An important approach to identifying amino acid residues within the hiding sites of enzymes is to exploit the specificity of the enzyme for a natural subs1ratc or regulatory ligand to limit chemical modification to the immediate vicinity of that site. In this approach. termed affinity labeling. a reagent is designed that is structurally similar to the normal ligand of the enzyme. but which also features a functional group capable of reacting covalently with many different amino acids. The expectation is that the affinity label will initially form a reversible enzyme-reagent complex analogous to the cyma-ligand complex and, once bound at the specific site, will react irreversibly with an amino acid residue within that site. The application o[ this technique can result in tagging of a particular binding site of a purified protein or in the labelling of one protein within a complex mixture of proteins. Affinity labeling can he used to localize a given site within a protein of knave amino acid sequence and thus to evaluate experimentally a site that was predicted on the basis of an amino acid sequence motif. It allows comparisons to be made between the substrate sites of the cyma in solution and those site localized within the crystalline form by X-ray diffraction. It can be used to test whether a compound bound irreversibly at a substrate site influences the enzyme conformation, subunit interaction, or the reactivity of other ligand site-,. Affinity labels with appropriate chromogenic or fluorescent substituents can be used to introduce spectral probes into specific sites on enzymes to report on the environment. on conformational changes, or on distances between functional sites. Affinity labelling can also provide the knowledge base to make rational choices of target amino acids for its directed mutagenesis. The general principles of affinity labelling have been summari1.ed in books and review articles (1-3).