Affinity label characteristics

Halomethyl ketones and acids are known to react with thiols and imidazoles. TPCK reacts far more rapidly with chymotrypsin than it does with normal histidine-containing peptides because of its high reactivity as an affinity label. This can be seen in Table 9.2 for an analogous chloromethyl ketone. In addition to this important diagnostic feature, the irreversible inhibition of chymotrypsin by TPCK has four other characteristic features 1,4... 

The activity falls off at low pH according to the ionization of a base of pKa 7, a characteristic value for a histidine residue His-57 is modified by the affinity label fos-L-phenylalanine chloromethyl ketone, with an irreversible loss of enzy-... 

A characteristic of an affinity label (R) is that it forms reversibly an enzyme-reagent complex (ER) prior to irreversible covalent modification to yield modified enzyme (ER ), as follows ... 

An essential characteristic of an affinity label is that it exhibits limited incorporation (ideally, I mol/mol active site) in causing inactivation. Indeed, the... 

Among the large number of site-specific reagents described in the literature, there are characteristics that are particularly desirable. In addition to structural similarity to the substrate or regulatory compound and the potential to react with many types of amino acids, these characteristics include water solubility, ease of synthesis, acceptable stability in aqueous solution over a moderate pH range, and the ability to form a stoichiometric, stable, isolable, and readily identifiable product from the reaction with enzyme. In this chapter, several chemical classes of affinity labels are considered, their notable features are evaluated in terms of these criteria, and selected examples of their application are presented. 

Extreme caution must be used in interpreting the experimental data obtained from studies of the site-specific modification of enzyme sites. For an enzyme whose structure has been determined by X-ray crystallography, such as bovine pancreatic ribonuclease, the results of affinity labeling by a reactive nucleoside can be compared with the crystal structures of various enzyme-ligand complexes (271). The general characteristics of the haloacetyl class of affinity labels have been summarized (281). 

Syncatalytic Enz3nne Modification Characteristic Features and Differentiation from Affinity Labeling... 

Factors Affecting Affinity Labeling. Pyruvate kinase incubated with DMPA for more prolonged periods of time is irreversibly inactivated. The characteristics of the inactivation process are as follows ... 

These approaches have all depended on one or more characteristics of individual transport systems that are not common to all systems the transport proteins must be removable from the membrane by osmotic shock, or be inducible, or retain binding activity upon solubilization from the membrane or removal by osmotic shock. An alternative that depends on a transport system having specificity for a particular substrate, or class of substrate, and high affinity for the substrate (s) is affinity labeling. A radioactive affinity label could, in principle, be used to specifically label the substrate binding proteins of a transport system and the label could be used as a marker to follow the purification of the protein. The experience gained could then be applied to isolation of the potentially active, unlabeled protein. 

Puromycin binds weakly and relatively unspecifically to ribosomes. Its use as an affinity probe is limited. Not unexpectedly, reactions of iV-iodoacetylpuromycin with ribosomes exhibit similar characteristics. On the other hand, a high binding constant does not necessarily guarantee a good affinity probe as exemplified by the streptomycin affinity labels. Vice versa, a compound with a weak binding constant might still be a superb affinity probe, since the outcome of experiments with halo-acetyl compounds depends so much on the presence of a properly oriented reactive amino acid side chain in the substrate binding site. This, of course, is a matter of chance. ... 

Affinity Labeling by Other Mechanisms. Tw o types of controls are useful to establish that covalent linking takes place from the photo-affinity-labeled base of tRNA and not from another residue. First, there should be no linking in the absence of photolysis. While this may appear to be a trivial control, we have in fact observed such a light-independent covalent reaction with NAG-tRNA at the ribosomal A site, which otherwise had all the characteristics of a specific reaction in that labeling required EF-Tu and poly(U), and no labeling w as observed from the ribosomal P site. 

---