Affinity label haloketones

The striking successes achieved by Shaw (1970a) and his coworkers with haloketone derivatives of N-tosyl-phenylalanine and a-N-tosyl-lysine as affinity labels for chymotrypsin and trypsin, respectively, have stimulated their use in a large number of affinity labels. Haloketones are potentially reactive with all the nucleophilic amino acid residues in proteins. Examples of residues modified by haloketones include methionine (Sigman et al. 1970), glutamate (Visser et al. 1971), cysteine (Porter et al. 1971), histidine (Schoellman and Shaw 1963) and serine (Schroeder and Shaw 1971). 

The synthesis of haloketones via their corresponding diazoketones is probably a more useful route for three reasons. They include 1) the fact that chloroketones as well as bromoketones can be readily prepared 2) the potential for oxidation of the affinity label by bromine is eliminated and 3) the radioactive form of the reagents can be readily synthesized using " C-diazomethane. The relevant synthetic route for the procedure is indicated in eq. (5.1). 

Amino acid derivatives formed by haloketones The identification of the amino acid residue modified by haloketones often represents a challenging problem. In fact, one inherent limitation in the use of most affinity labels is that authentic samples of the various amino acid derivatives formed are not readily available since the syntheses from the free amino acids and the affinity label are not simple. 

The derivatives of methionine formed by haloketones are not stable to the usual conditions of acid hydrolysis. These sulfonium salts are degraded in three different ways. Some methionine is regenerated, some homoserine and homoserine lactone is formed and possibly the homocysteine derivative of the general structure indicated below is produced where X represents the rest of the affinity label. 

Amides and esters of haloacids have been frequently used in the synthesis of affinity labels. Like haloketones, these derivatives react with all the nucleophilic amino acids. Their advantages as modification reagents are two-fold. First, they are relatively more easy to synthetize than haloketones. Any substrate or reversible inhibitor with a free amino or hydroxyl group can be potentially converted into an affinity labelling reagent. Second upon acid hydrolysis, the modified protein yields carboxymethylated derivatives which are known and therefore readily identifiable and quantifiable. 

Since epoxides potentially will react with a variety of amino acids, many products may be formed if they are incorporated into affinity labels. However at present affinity labels containing epoxides have been shown to modify either glutamate or aspartate residues in pepsin, lysozyme, those phosphate isomerase and j -glucosidase. The only other residue which has as yet been modified by epoxides is methionine 192 of chymotrypsin. In general, the other possible products should be similar in stability to derivatives formed by haloketones. Therefore the methods for identification of the amino acid derivatives formed by reaction with epoxides closely parallel those described in connection with haloketones ( 5.3.2). 

One of the first affinity labels described was the chloromethylketone derived from A -tosyl-L-phenylalanine (TPCK), which provided convincing evidence for the catalytic involvement of an imidazole side chain in proteolytic enzymes. This and other successes of Elliott Shaw > and his colleagues stimulated many investigators to design ligandlike a-haloketones as probes of the active sites of many different enzymes and nonenzymic proteins. a-Haloketones remain one of the more popular chemical classes of affinity labels. 

Successful application of affinity labeling, with haloketones or any other class of reagent, typically requires four types of endeavors (1) search for suitable reagents (2) demonstration that inactivation results from an active-site-directed process (3) characterization of the covalently modified protein (4) determination of the relationship of the results to the mechanism of the enzyme-catalyzed reaction. 

Most of the methodologies used and problems encountered with halo-ketones as affinity labels are covered by considerations of bromopyruvate, haloacetol phosphates, and haloketone derivatives of pyridine nucleotides. Similar information is gained from numerous elegant studies in which halomethylketone derivatives of amino acids and peptides have been used as affinity labels for proteases. These investigations are excluded from the present article, since they are considered elsewhere in this volume. ... 

The most successful application of haloacetol phosphates as affinity labels has been in the partial characterization of the active site of triosephosphate isomerase. Very similar studies, carried out independently in the laboratories of F. C. Hartman and J. R. Knowles, demonstrated an essential glutamyl y-carboxylate (esterified by the reagent) in the enzyme. The recently determined primary structure of the enzyme from rabbit muscle places the glutamyl at position 165. All the usual criteria of affinity labeling were satisfied and have been well documented. Certain aspects of these studies that either relate to the use of haloketones in general or have provided evidence of the carboxylate s intimate role in the catalytic process will be considered. 

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