Esterase reaction

F. M. Dickinson, G. W. Haywood, The Effects of Mg2+ on Certain Steps in the Mechanism of the Dehydrogenase and Esterase Reactions Catalysed by Sheep Liver Aldehyde Dehydrogenase. Support for the View That Dehydrogenase and Esterase Activities Occur at the Same Site on the Enzyme , Biochem. J. 1986, 233, 877-883. 

Some other reactions, such as aldehyde hydration (29) and e ter hydrolyses (30—33) are also catalyzed by the enzyme, but much j ess efficiently than the reversible hydration of CO 2. The esterase reaction, in particular, has been very useful in the kinetic analysis of carbonic anhydrase function, however. 

Zinc can be removed from carbonic anhydrase on dialysis against a chelating agent at pH about 5 (37, 38). The apoenzyme is inactive but the gross conformation of the protein is maintained (37, 39, 40). The metal-chelating site can accomodate any of the divalent transitional metal ions from Mn2+ to Zn2+ as well as Cd2+ and Hg2+ (38,41). Most of these metallocarbonic anhydrases have low activities or are inactive, however. Only Zn2+ and Co2+ are efficient activators. As shown in Table 3, this narrow metal-ion specificity is observed for the CO 2 hydration as well as for the esterase reactions. 

Although NAD inhibits the esterase reaction, adenine nucleotides stimulate it, which suggests that NAD is blocking access of the aromatic ester to the reactive thiol in a manner similar to its effect on iodoaceta-mide alkylation (70). Acyl shifts involving Cys-149 and Lys-183 also occur readily with p-nitrophenyl acetate at higher pH and in the absence of NAD (70, 73, 74). 

At pH values around 6 yn 7.5, the inorganic phosphate (pXa = 6.82) exists as a mixture of HO-POsH and HO-PO forms. Similarly, the terminal phosphates of ATP (pKa = 6.95) and ADP (pS — 6.68) exist as mixtures of the mononegative and dinegative ions. It would be much simpler to define in terms of total [ATP], [ADP], and [P ]- bet us return to our esterase reaction in order to see how total analytical concentrations are incorporated into the Xiq and AG expressions. We would like to determine the value for KL, where ... 

Figure 1 The native reaction of carbonic anhydrase (CO2 hydration, top) and its promiscuous aryi esterase reaction, exempiified with naphthyi acetate (bottom). Both reactions proceed by the same mechanism of hydroxide ion attack on a carbonyi, foiiowed by the stabiiization of an oxyanion intermediate by the active-site Zn +. Despite this obvious simiiarity, the EC numbers of these reactions differ in the first digit (T ble 1, entry 15).

Greenzaid, P. Jenks, W.P. (1971) Pig liver esterase. Reactions with alcohols, structure-reactivity correlations, and the acyl-enzyme intermediate. BiochemistrytO, 1210. 

Regioselectively protected cytidine derivatives have been prepared from the peracetylated nucleoside using lipase and esterase reactions. There have been reports on the use of bacterial proteases for the selective acylation of sucrose to produce a variety of different acyl and acrylate esters. 8-Aminooctyl 5-5-coniferyl-5-thio-a-L-arabinofuranoside(27) attached to sepharose proved to be a selective affinity ligand for feruloyl esterase A. niger ... 

The Zn(ii) complex (2.144) mimics the esterase reaction, as shown in Scheme 2.23. The complex uses lariat azacrown ethers for the hydrolysis of ester groups. This is of significance interest because of the hydrolysis of phosphate... 

Tris buffer pH 8 was used in the esterase reactions and phosphate buffer pH 7 was used in the lipase reactions. 5% DMSO or MeOH was aiso used in all the reactions to increase the solubility of 4 in aqueous buffer. The substrate concentration was 2.2 mM. Cat., catalyst. 

Nomenclature. The compound on which the enzyme acts is known as the substrate. The name of the enzyme is now usually obtained by adding the termination ase to the name of the substrate. Thus an enzyme which hydrolyses an ester is known as an esterase. Nevertheless the older names of many enzymes still persist owing to their early disco ieiy. In some cases the name of the enzyme indicates the reaction w hich it catalyses, e.g. oxidase. 

One approach called enzymatic resolution, involves treating a racemic mixture with an enzyme that catalyzes the reaction of only one of the enantiomers Some of the most commonly used ones are lipases and esterases enzymes that catalyze the hydrol ysis of esters In a typical procedure one enantiomer of the acetate ester of a racemic alcohol undergoes hydrolysis and the other is left unchanged when hydrolyzed m the presence of an esterase from hog liver... 

Immobilization. The fixing property of PEIs has previously been discussed. Another appHcation of this property is enzyme immobilization (419). Enzymes can be bound by reactive compounds, eg, isothiocyanate (420) to the PEI skeleton, or immobilized on soHd supports, eg, cotton by adhesion with the aid of PEIs. In every case, fixing considerably simplifies the performance of enzyme-catalyzed reactions, thus faciHtating preparative work. This technique has been appHed to glutaraldehyde-sensitive enzymes (421), a-glucose transferase (422), and pectin lyase, pectin esterase, and endopolygalacturonase (423). 

The reaction between esterase and phosphorus inhibitor (109) is bimolecular, of the weU-known S 2 type, and represents the attack of a nucleophilic serine hydroxyl with a neighboring imida2ole ring of a histidine residue at the active site, on the electrophilic phosphorus atom, and mimics the normal three-step reaction that takes place between enzyme and substrate (reaction ). 

Dica.rboxyIic AcidMonoesters. Enzymatic synthesis of monoesters of dicarboxyUc acids by hydrolysis of the corresponding diesters is a widely used and thoroughly studied reaction. It is catalyzed by a number of esterases. Upases, and proteases and is usually carried out in an aqueous buffer, pH 6—8 at room temperature. Organic cosolvents may be added to increase solubiUty of the substrates. The pH is maintained at a constant level by the addition of aqueous hydroxide. After one equivalent of base is consumed the monoesters are isolated by conventional means. 

Optically Active Acids and Esters. Enantioselective hydrolysis of esters of simple alcohols is a common method for the production of pure enantiomers of esters or the corresponding acids. Several representative examples are summarized ia Table 4. Lipases, esterases, and proteases accept a wide variety of esters and convert them to the corresponding acids, often ia a highly enantioselective manner. For example, the hydrolysis of (R)-methyl hydratropate [34083-55-1] (40) catalyzed by Hpase P from Amano results ia the corresponding acid ia 50% yield and 95% ee (56). Various substituents on the a-carbon (41—44) are readily tolerated by both Upases and proteases without reduction ia selectivity (57—60). The enantioselectivity of many Upases is not significantly affected by changes ia the alcohol component. As a result, activated esters may be used as a means of enhancing the reaction rate. 

Optically Active Alcohols and Esters. In addition to the hydrolysis of esters formed by simple alcohols described above, Hpases and esterases also catalyze the hydrolysis of a wide range of esters based on more complex and synthetically useful cycHc and acycHc alcohols (Table 5). Although the hydrolysis of acetates often gives the desirable resolution, to achieve maximum selectivity and reaction efficiency, comparison of various esters is recommended. 

Chirazymes. These are commercially available enzymes e.g. lipases, esterases, that can be used for the preparation of a variety of optically active carboxylic acids, alcohols and amines. They can cause regio and stereospecific hydrolysis and do not require cofactors. Some can be used also for esterification or transesterification in neat organic solvents. The proteases, amidases and oxidases are obtained from bacteria or fungi, whereas esterases are from pig liver and thermophilic bacteria. For preparative work the enzymes are covalently bound to a carrier and do not therefore contaminate the reaction products. Chirazymes are available form Roche Molecular Biochemicals and are used without further purification. 

An ester of alanine with an arylaliphatic alcohol has shown promise as a non-tricyclic antidepressant. It may be speculated that the hindered milieu of the ester linkage protects the compound from hydrolysis by endogenous esterases. The preparation starts by reaction of pheny-lacctate 83 with methyl magnesium iodide to give tertiary carbinol 84. Acylation with 2-bromo-]>ropionyl bromide leads to ester 85 displacement of halogen with ammonia leads to alaproclate ( 6) [211. 

Specificity is not always perfect. Sometimes an enzyme will work with any member of a class of compounds. For example, some esterases (enzymes that catalyze the reaction of esters with water) will work with numerous esters of similar, but different, structures. Usually, in cases of this kind, one of the members of the substrate class will react faster than the others, so the rates will vary from one substrate to another. 

These are major disadvantage of the esterase resolution process. Since die optimum pH of die enzymic reaction is generally on the alkaline side, die esters used as substrates are non-enzymatically hydrolysed and die optical purity of die L-amino adds obtained is generally low. Also the substrate has to be protected at the amino group in most cases in order to prevent formation of diketopiperasines. The esterase method is not attractive in practice and to the best of our knowledge is not used on an industrial scale. 

Estane VC products, 201 Ester-amide copolymers, 146, 147 Esterases, 82 Esterification, direct, 63 Ester interchange catalysts, 71 Ester interchange reactions, 31, 62-63,... 

Esterases, proteases, and some lipases are used in stereoselective hydrolysis of esters bearing a chiral or a prochiral acyl moiety. The substrates are racemic esters and prochiral or meso-diesters. Pig liver esterase (PLE) is the most useful enzyme for this type of reaction, especially for the desymmetrization of prochiral or meso substrates. 

The principal methods for the hydrolase-promoted synthesis of enantiomerically pure alcohols are depicted in Figure 6.44. Biocatalytic acylation and alcoholysis have been reviewed recently [116,117]. Lipases, esterases, and proteases catalyze these reactions, but CAL-B [118-120], CRL [121,122], and diverse lipase preparations from Pseudomonas species are common place. 

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