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Site-specific modification of enzyme sites

Colman, R. F. (1990). Site-Specific Modification of Enzymes Sites. The Enzymes. 19, San Diego, Academic Press. 283-321. [Pg.192]

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). [Pg.312]

Colman has also produced a definitive account of site-specific modification of enzymes, and her chapter is particularly instructive about the range and utility of reaction types that can be gainfully exploited in affinity labeling experiments. [Pg.39]

The site-specific modification of enzymes with a single electron-relay group located near to the redox cofactor and providing efficient electrical contact with the conductive support has been achieved by the reconstitution of enzymes with cofactors covalently linked to redox groups. Affinity interactions between enzymes and their cofactors at the electrode interface can allow the efficient electrical contacting of aligned proteins. [Pg.2526]

The application of site-specific modification of enzymes and other proteins has become increasingly common, and a wide range of chemical classes is available from which to select or design a reagent for exploring a particular enzyme. Structural similarity to the natural ligand is always desirable to ensure target sped-... [Pg.313]

To date (ca 1996) many potentially usefiil sucrose derivatives have been synthesized. However, the economics and complexities of sucrochemical syntheses and the avadabiLity of cheaper substitutes have limited their acceptance hence, only a few of them are in commercial use. A change in the price and availability of petroleum feedstocks could reverse this trend. Additional impetus may come from regioselective, site-specific modifications of sucrose to produce derivatives to facilitate and improve the economics of sucrochemical syntheses. For example, the microbe yigwbacterium tumifaciens selectively oxidizes sucrose to a three-keto derivative, a precursor of alkylated sucroses for detergent use (50). Similarly, enzymes have been used for selective synthesis of specific sucrose derivatives (21). [Pg.6]

The principle advantage of the physical labeling method is the possibility of receiving direct information about the structure, mobility and local micropolarity of certain parts of a molecular object of any molecular mass. Developments in synthetic chemistry, biochemistry and site-directed mutagenesis have provided researchers with a wide assortment of labels and probes, and have paved the way for the specific modification of protein function groups, including enzyme active sites. [Pg.133]

Site-specific sulfenylation of tryptophan residues in egg-white lysozyme has been attained by either limiting the amount of reagent, or modifying the reaction conditions. Thus, Trp-108 is the major site of modification upon addition of one equivalent of 2-thio-(2-nitro-4-carboxyphenyljsulfenyl chloride to lysozyme in 25% acetic acid (Veronese et al. 1972). Specific modification of Trp-62 was attained in approximately 8 hr by the addition of 5 consecutive portions of solid NPS-Cl (10 /imole for each ml of reaction mixture) to a solution of lysozyme (0.5 /imole/ml) in 0.1 M sodium acetate at pH 3.5 (Shechter et al. 1972). In both cases, the protein derivative was separated from other products, and unreacted enzyme by ion-exchange chromatography. [Pg.94]

Two factors are chiefly, but not exclusively, responsible for the fact that, under certain conditions, amino acids in native proteins react more rapidly than free amino acids in solution. The first and most general is the capacity of proteins to bind modification reagents at or near the functional groups of amino acid residues in orientations favorable to reaction. The reversible binary complexes formed between proteins and modification reagents prior to reaction are analogous to enzyme-substrate complexes. As a result, most site-specific modifications of native proteins probably proceed by the scheme summarized in eq. [Pg.123]

Reactivity is not limited to direct addition to the phenoxyl ring structure, and reactions with thiols, amines and olefmic compounds have been reported. Both HOBr and HOCl can elicit oxidation of cysteine residues, where oxidation of the thiol moiety to a sulphinic acid is associated with activation of the latent form of matrix metalloproteinase-7 [111]. Higher doses of HOCl cause inactivation of the enzyme through site-specific modification of tryptophan and the adjacent glycine to yield an unknown product that lacks four mass units [112]. [Pg.55]

Circular dichroism (CD) has played an important role in our studies on the modification of enzymes and hormones with Co(III). The objective of these studies has been to incorporate selectively substitution inert metal ions at specifically modified sites in proteins as probes of biological function. Significant information concerning the catalytic mechanism of carboxypeptidase A (CPA) (1) has been obtained from a site specific modification of tyrosine 248 with Co(III) (2). The method developed for CPA has been extended to other enzymes and hormones in order to devg op an improved method for incorporating stable radioisotopes t Co) into proteins. The substitution-inertness of Co(III) provides the necessary stability in these derivatives (3). [Pg.195]

Figure 4 Strategies for the application of CoA analogues. For in vitro studies appropriate analogues are prepared from a suitable precursor, purified, and characterized if necessary, and then normally used as mechanism-based probes and inhibitors (panel A). When CoA analogues are used for the in vitro site-specific modification of proteins by transfer of a reporter label to a carrier protein module using a PPTase enzyme, the analogue can be prepared and used in situ (panel B). In vivo reporter labeling is made possible when cells are provided with suitably modified pantothenamide precursors, which are subsequently transformed into the respective CoA analogues and transferred a carrier protein by the cell s native CoA biosynthesis (CoaADE) and PPTase enzymes. The labeled protein can be recovered from the cells by cell lysis. Figure 4 Strategies for the application of CoA analogues. For in vitro studies appropriate analogues are prepared from a suitable precursor, purified, and characterized if necessary, and then normally used as mechanism-based probes and inhibitors (panel A). When CoA analogues are used for the in vitro site-specific modification of proteins by transfer of a reporter label to a carrier protein module using a PPTase enzyme, the analogue can be prepared and used in situ (panel B). In vivo reporter labeling is made possible when cells are provided with suitably modified pantothenamide precursors, which are subsequently transformed into the respective CoA analogues and transferred a carrier protein by the cell s native CoA biosynthesis (CoaADE) and PPTase enzymes. The labeled protein can be recovered from the cells by cell lysis.
Specific modification of Cys-46. Li and Vallee 86,87) and Harris 86) found that one cysteine residue per subunit may be selectively carboxymethylated with iodoacetate. The modified enzyme is inactivated and this cysteine residue, Cys-46 92), was suggested to be at the active site of the enzyme. The same residue in the S subunit is also especially reactive 20,94). The modification is preceded by anion binding of the iodoacetate and stimulated by the presence of imidazole 140,142,142). By using these facts and working with the crystalline enzyme, it is possible to achieve a highly specific and complete modification (ISO). X-ray studies of the carboxymethylated enzyme and the reaction mechanism of this modification are described in Section II,H. The carboxymethylation has been used to establish that both the EE 19) and SS 20) isozymes are active in u oxidations of fatty acids. [Pg.142]


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Enzymic modification

Modification of enzyme

Site specificity

Specificity of enzymes

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