Active-site-directed chemical

ABPP) applies active site-directed chemical probes to measure dynamics in enzyme activities, directly in the context of whole proteomes and living systems. 

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

Activity-based protein profiling (ABPP) is a chemical proteomic strategy in which active-site-directed covalent probes are used to profile the functional states of enzymes in complex proteomes. Activity-based probes (ABPs) can distinguish active enzymes from their inactive zymogens or inhibitor-bound forms. They contain a reactive group intended to modify enzyme active sites covalently and a reporter group (typically rhodamine or biotin) that assists in detection and identification of protein targets. 

The most promising tools developed for this sort of analysis are active-site-directed irreversible inhibitors of DUBs. These inhibitors are ubiquitin or ubiquitin-like proteins chemically modified at the C-terminus by an electrophilic moiety such as a Michael acceptor or alkyl halide. The modified ubiquitin can be incubated with a purified DUB or a cell lysate containing DUB activity. Ubiquitin vinyl sul-fone (UbVS) is one such irreversible inhibitor because the vinyl sulfone moiety reacts with the active-site cysteine of the DUB, forming a thioether linkage. The covalent adduct is stable and can be detected in a variety of ways. Labeling of DUBs is specific, as only a DUB active-site cysteine will efficiently react with the vinyl sulfone moiety. 

The procedure to phosphorylate riboflavin derivatives on a preparative scale has recently been improved . These preparations, and also commercial FMN, contain a considerable amount of riboflavin phosphate isomers, which are difficult to separate by column chromatography. This problem is emphasized in the chemical synthesis of FAD where the yield is rather low (20-25 %). In this context, it is surprising that a modification of the synthesis of FAD from FMN published by Cramer and Neuhoeffer has not been noticed by workers in the flavin field. According to Cramer and Neuhoeffer, the yield of the chemical synthesis of FAD is drastically improved ( 70 % pure FAD). The procedure was successfully applied in the author s own laboratory (yield 60-70%). It is expected that the improved procedure of the FAD synthesis will stimulate the active-site directed studies on flavoproteins because the problem of separating FMN or FAD from their synthetic by-products has already been solved by use of FMN- or FAD-specific affinity column... 

An affinity label, or active-site-directed irreversible inhibitor, is a chemically reactive compound that is designed to resemble a substrate of an enzyme, so that it binds specifically to the active site and forms covalent bonds with the protein residues.1-3 Affinity labels are very useful for identifying catalytically important residues and determining their pKa values from the pH dependence of the rate of modification. 

Chemical Modification by Active-Site-Directed Reagents Elliott Shaw... 

One of the first interesting structures with cholinergic properties isolated from a marine source is the chemical onchidal. This compound was first isolated from the mollusc Onchidella binneyi and has an acetate ester similar to acetylcholine. Upon isolation, onchidal was discovered to be an active site-directed irreversible inhibitor of AChE (Abramson et ah, 1989). 

Recent achievements in the development of active-site directed affinity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (24-27). These specific activity probes allow joint evaluation of selective protease inhibition concomitant with labeling of relevant protease enzymes for more analyses. Moreover, activity-based probes that selectively label the main protease subclasses—cysteine, serine, metallo, aspartic, and threonine—can provide advantageous chemical approaches for functional protease identification. Activity probe labeling of proteases allows direct identihcation of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identification of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article. 

Although the organic modifiers are usually not specific for a given enzyme, the second group, the affinity labels, have a degree of specificity built in. Sometimes described as active-site directed, irreversible inhibitors, affinity labels are usually substrate or product analogs that contain an additional chemically reactive moiety. They first bind to the en-... 

E. Shaw inP. D. Boyer, Ed, Chemical Modification by Active-Site Directed Reagents, Acch demic Press, New York, 191Q,pp. 91-147. 

Studies on the inactivation mechanism of MAO by active site-directed reagents are potentially useful in defining the chemical mechanism of the normal reaction. In this review, only compounds known to be mechanism-based inhibitors will be studied, particularly in their relationship to elucidating the chemical mechanism of this enzymic redox reaction. 

Active-site modifications. Chemical modification and site-directed mutagenesis experiments suggest that Glu-270 is essential for cataly-... 

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