Affinity labels amino acid selective

Table 8.3 Examples of amino acid selective affinity labels...

An affinity label is a molecule that contains a functionality that is chemically reactive and will therefore form a covalent bond with other molecules containing a complementary functionality. Generally, affinity labels contain electrophilic functionalities that form covalent bonds with protein nucleophiles, leading to protein alkylation or protein acylation. In some cases affinity labels interact selectively with specific amino acid side chains, and this feature of the molecule can make them useful reagents for defining the importance of certain amino acid types in enzyme function. For example, iodoacetate and A-ethyl maleimide are two compounds that selectively modify the sulfur atom of cysteine side chains. These compounds can therefore be used to test the functional importance of cysteine residues for an enzyme s activity. This topic is covered in more detail below in Section 8.4. 

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. 

The most interesting class of protease inhibitors in viral studies has been the chloromethyl ketone derivatives of amino acids (54j 55) These were designed as affinity labels of serine-type proteases, and react irreversibly with histidine and serine residues in the active sites of proteases. There is a basis for selectivity of the chloromethyl ketones phenylalanyl and lysyl derivatives were synthesized, which had specificity for chymotrypsin and trypsin, respectively (54) This specificity led to studies on inhibition of poliovirus protein cleavage, with positive results (25, 26). 

In the BONCAT method, the cellular system of interest (cultured cells, tissue slices, or live animals) is pulse-labeled with a non-canonical amino acid that carries a reactive side chain. In our initial experiments, we used azidohomoalanine (Aha) as the label because Aha-Iabeled proteins can be selectively tagged with dyes or affinity reagents through copper-catalyzed or strain-promoted azide-alkyne cycloaddition reactions [34-36]. Tagged proteins can then be separated from other... 

Suicide Substrates. Much like affinity labels, suicide inhibitors first form a reversible complex with the target enzyme due to the structural similarity between inhibitor and substrate. In a subsequent time dependent step, an irreversible complex (usually covalent) is formed with an appropriately positioned amino acid side chain. Unlike the affinity label, suicide substrates (65) are not inherently reactive and must undergo activation by the target enzyme before the irreversible complex is formed. Therefore, these inhibitors are generally more selective than affinity labels. Since the enzyine catalyzes its own inactivation, these inhibitors are also known as kcat inhibitors (66), enzyme-activated, irreversible inhibitors (54) and Trojan horse reagents (6. ... 

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