CENTA, a chromogenic cephalosporin, is readily hydrolyzed by ␤-lactamases of all classes except for the Aeromonas hydrophila metalloenzyme. Although it cannot practically be used for the detection of ␤-lactamaseproducing strains on agar plates, it should be quite useful for kinetic studies and the detection of the enzymes in crude extracts and chromatographic fractions.Nitrocefin and, to a lesser extent, PADAC have been used as chromogenic substrates of ␤-lactamases. Such substrates, whose hydrolysis can be directly monitored in the visible wavelength range, are of particular interest for the kinetic characterization of ␤-lactamases. Nitrocefin has, for instance, been widely used as a reporter substrate in the study of the inactivation of ␤-lactamases or of their interactions with poor substrates (3). It also allows the rapid identification of active fractions during ␤-lactamase purification. However, the price of nitrocefin has recently been increased significantly, and PA-DAC is no longer commercially available. Synthesis of compounds is also rather tedious. It is thus surprising that a third chromogenic cephalosporin, CENTA ( Fig. 1), which can be prepared from the commercially available drug cephalothin, has not received more attention, although it was shown to be sensitive to many ␤-lactamases (11). In the study described in this report, we determined the kinetic parameters characterizing the interactions between CENTA and a representative set of ␤-lactamases and some penicillin-binding proteins (PBPs). Although CENTA cannot be used for the direct detection of ␤-lactamase-producing colonies on agar plates, it still represents an interesting alternative to nitrocefin for the kinetic characterization of ␤-lactamases.CENTA was prepared as follows: 3-carboxyl-4-nitrothiophenol (TNB) was obtained by dissolving 5.05 mmol (2 g) of 5,5Ј-dithio-bis-(2-nitrobenzoic acid) in 100 ml of an aqueous solution of 0.5 M Tris base, and the pH was adjusted to 8.0 by addition of 6 M HCl. Dithiothreitol (7.1 mmol, 1.1 g) was added, and the solution turned orange-red. The mixture was stirred for 10 min at 22°C and extracted six times with 25 ml of ethyl acetate before being acidified to pH 1.5 by addition of 6 M HCl. The residual ethyl acetate was eliminated by bubbling nitrogen through the solution, which was thereafter left overnight at 4°C. The precipitate (TNB) was collected by filtration, washed, and dried. The sodium salt of cephalothin (1 g, 2.4 mmol) and 1 equivalent of TNB (478 mg) were dissolved in 19 ml of H 2 O, and the pH was adjusted to 7.0 with 1 M NaOH. The solution was stirred for 6 h at 65°C. The cooled solution was extracted with 10 ml of ethyl acetate, acidified to pH 2.0 with 1 M HCl, and extracted three times with 15 ml of ethyl acetate. The organic phase was washed three times with 15 ml of water, dried over MgSO 4 , and evaporated to dryness in vacuo. The sodium salt of CENTA was obtained by dissolving the dry residue in 25 ml of water containing 1 equivalent of NaHCO 3 , and the solution was freeze-dried,...

It is shown that anilide substrates of chymotrypsin may bind in a nonproductive mode, with the anilide ring in the substrate binding pocket. Acetyl-and trimethylammoniumsubstituted TV-acetyl-u-tyrosineanilides bind predominantly productively. Hydrophobic substituents such as -Cl and -NO% groups cause nonproductive binding to be 50-100 times stronger than productive. The tight binding of these hydrophobic substrates is not due to the accumulation of a covalent intermediate. • Similarly, the lowered ¿cat values are not due to electronic effects on chemical reactivity but are a consequence of the nonproductive binding. At pH 7.8 the dissociation constants of V-acetyl-L-tyrosine-4-chloroanilide, V-acetylglycine-4-chloroanilide, and formyl-4-chloroanilide are 0.78, 0.98, and 0.40 mM, respectively. The binding is almost completely associated with that of the 4-chloroanilide portion. The dissociation constants of a series of substituted formanilides follow 7r, the hydrophobicity constant. The binding of substituted A-acetyltyrosineanilides can be separated into the T A he chymotrypsin-catalyzed hydrolysis of specific anilide substrates is of some mechanistic and historical importance.Substituents in the aniline ring cause inductive effects which alter chemical reactivity but, it would be thought, cause only small steric effects. The hydrolysis of a series of substituted anilides of TV-acetyl-L-tyrosine and V-acetyl-L-tryptophan has led to three theories of the mechanism of action of chymotrypsin based on the experimental linear free-energy relationships.It was found that for a limited series of substituted anilides both ¿cat* 1 **and Km are decreased by electron withdrawal and that the apparent pKa for the enzyme-substrate complex is in some cases significantly lowered (Inagami et al., 1969). These authors suggested the possible contribution of an acidic group in the catalysis. Wang and coworkers (Wang and Parker, 1968; Parker and Wang, 1969) have formulated a general theory of enzyme catalysis by "directed proton transfers" based on the variation of ¿cat with aniline basic strength. Caplow (1969) and Lucas and Caplow (1972), on the basis of the variation of ¿cat, Km, and the of ¿cat with substituents, postulated the accumulation of a tetrahedral intermediate in the reaction-the enzyme stabilizing the otherwise unstable intermediate.

An attractive hypothesis in enzyme evolution relies on the promiscuous activities that already exist in enzymes, which can be used as pipelines for the selection of new catalytic activities. [1] These new activities are usually low, but can serve as starting points in Darwinian evolution. In metalloenzymes, the diversity of promiscuous activity is increased by the variety of metallic ions that can be incorporated in the active site and can catalyse a wide range of chemical transformations. As an example, the iron-containing rubredoxin oxidase and zinc-b-lactamase are two phylogenetically related enzymes that catalyse oxidation and hydrolysis reactions with unrelated mechanisms. [2] This principle could be applicable in the field of directed evolution as a way to discover primitive artificial metalloenzymes that might catalyse challenging asymmetric transformations. In this work, we replaced the zinc ion of natural carbonic anhydrase with manganese and showed that the resulting product can catalyse the enantioselective epoxidation of styrene.Protein modification with cofactors or metal-containing complexes is an approach that has been widely used for the generation of new catalytic activities, [3] but few examples in the field of asymmetric catalysis have yet appeared.[4] The very first was reported in 1978 by Wilson and Whitesides, on the incorporation of a biotinylated achiral rhodium diphosphine complex in avidin for the hydrogenation of dehydroamino acids; [5] this concept has recently been improved through combination of structural variants of the biotinylated complex with several wild-type and mutant proteins.[6] A similar noncovalent approach was used for the enantioselective oxidation of sulfides after incorporation of a vanadate ion into a hydrolase A C H T U N G T R E N N U N G homologous to vanadium-dependent peroxidases [7] or of an A C H T U N G T R E N N U N G achiral chromium salophen complex into apomyoglobin.[8] Covalent grafting of achiral organometallic complexes has also been used as a complementary approach, with attachment of a copper phenanthroline complex to the adipocyte lipid-binding protein affording enantioselective hydrolysis of esters and amides.[9] More recently, the dual anchoring of an achiral manganese salen complex in apomyoglobin for asymmetric sulfoxidation has been reported. [10] In all these approaches the metallic ion is part of a larger achiral complex or ion and the chiral environment is provided by the protein.Our approach consists of incorporating only the catalytic metal ion in a protein featuring a suitable site for coordination, taking advantage of an attractive recently reported procedure for the manganese-catalysed epoxidation of alkenes.[11] This method uses hydrogen peroxide in hydrogen carbonate buffer, and the authors postulated the formation of peroxymonocarbonate-HCO 4 À -as the actual oxygen-transfer reagent. We selected carbonic anhydrase as a suitable candidate for hosting the manganese ion in its active site because the active species of the epoxidation...

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