Ester formation, protease

Lipases are the enzymes for which a number of examples of a promiscuous activity have been reported. Thus, in addition to their original activity comprising hydrolysis of lipids and, generally, catalysis of the hydrolysis or formation of carboxylic esters [107], lipases have been found to catalyze not only the carbon-nitrogen bond hydrolysis/formation (in this case, acting as proteases) but also the carbon-carbon bond-forming reactions. The first example of a lipase-catalyzed Michael addition to 2-(trifluoromethyl)propenoic acid was described as early as in 1986 [108]. Michael addition of secondary amines to acrylonitrile is up to 100-fold faster in the presence of various preparations of the hpase from Candida antariica (CAL-B) than in the absence of a biocatalyst (Scheme 5.20) [109]. 

As discussed above, proteases are peptide bond hydrolases and act as catalysts in this reaction. Consequently, as catalysts they also have the potential to catalyze the reverse reaction, the formation of a peptide bond. Peptide synthesis with proteases can occur via one of two routes either in an equilibrium controlled or a kinetically controlled manner 60). In the kinetically controlled process, the enzyme acts as a transferase. The protease catalyzes the transfer of an acyl group to a nucleophile. This requires an activated substrate preferably in the form of an ester and a protected P carboxyl group. This process occurs through an acyl covalent intermediate. Hence, for kineticmly controlled reactions the eii me must go through an acyl intermediate in its mechanism and thus only serine and cysteine proteases are of use. In equilibrium controlled synthesis, the enzyme serves omy to expedite the rate at which the equilibrium is reached, however, the position of the equilibrium is unaffected by the protease. 

Fig. 3 Mechanisms for enzymatic supramolecular polymerisation (a) Formation of supramolecular assembly via bond cleavage, (b) Formation of supramolecular assemblies via bond formation. Examples are shown of biocatalytic supramolecular polymerisation of aromatic peptide amphiphiles via (i) phosphate ester hydrolysis, (ri) alkyl ester hydrolysis, and (iii) amide condensation or reversed hydrolysis using protease...

Formation of an amide bond (peptide bond) will take place if an amine and not an alcohol attacks the acyl enzyme. If an amino acid (acid protected) is used, reactions can be continued to form oligo peptides. If an ester is used the process will be a kinetically controlled aminolysis. If an amino acid (amino protected) is used it will be reversed hydrolysis and if it is a protected amide or peptide it will be transpeptidation. Both of the latter methods are thermodynamically controlled. However, synthesis of peptides using biocatalytic methods (esterase, lipase or protease) is only of limited importance for two reasons. Synthesis by either of the above mentioned biocatalytic methods will take place in low water media and low solubility of peptides with more than 2-3 amino acids limits their value. Secondly, there are well developed non-biocatalytic methods for peptide synthesis. For small quantities the automated Merrifield method works well. 

The currently accepted mechanism for the hydrolysis of amides and esters catalyzed by the archetypal serine protease chymotrypsin involves the initial formation of a Michaelis complex followed by the acylation of Ser-195 to give an acylenzyme (Chapter 1) (equation 7.1). Much of the kinetic work with the enzyme has been directed toward detecting the acylenzyme. This work can be used to illustrate the available methods that are based on pre-steady state and steady state kinetics. The acylenzyme accumulates in the hydrolysis of activated or specific ester substrates (k2 > k3), so that the detection is relatively straightforward. Accumulation does not occur with the physiologically relevant peptides (k2 < k3), and detection is difficult. 

Peptide a-oxo acids 1 (R4=H), a-oxo esters 1 (R4= alkyl or substituted alkyl), and a-oxo-amides 2 (R5=R6=H, alkyl, substituted alkyl, aryl, and/or heteroaryl) are potent reversible inhibitors for cysteine and serine proteases (Scheme 1).[1 9 Their inhibitory potency is the result of their enhanced electrophilic a-carbonyl functional group that can better compete with the substrate in the formation of a tetrahedral adduct with the cysteine or serine residue at the protease active site. In the case of peptide a-oxo esters and a-oxoamides, the extension in PI and beyond gives the inhibitors additional interactions with the protease at the corresponding sites. 

The trypsin family of serine proteases includes over 80 well-characterized enzymes having a minimum sequence homology of >21%. Two amino acid residues are absolutely conserved (Cysl82, Glyl96) within their active sites [26,27]. These proteases have similar catalytic mechanisms that lead to hydrolysis of ester and amide bonds. This occurs via an acyl transfer mechanism that utilizes proton donation by histidine to the newly formed alcohol or amine group, dissociation and formation of a covalent acyl-enzyme complex. 

Recently a simplified process was developed for incorporating l-methionine directly into soy proteins during the papain-catalyzed hydrolysis (21). The covalent attachment of the amino acid requires a very high concentration of protein and occurs through the formation of an acyl-enzyme intermediate and its subsequent aminolysis by the methionine ester added in the medium. From a practical point of view, the main advantage of enzymatic incorporation of amino acids into food proteins, in comparison with chemical methods, probably lies in the fact that racemic amino acid esters such as D,L-methionine ethyl ester can be used since just the L-form of the racemate is used by the stereospecific proteases. On the other hand, papain-catalyzed polymerization of L-methio-nine, which may occur at low protein concentration (39), will result in a loss of methionine because of the formation of insoluble polyamino acid chains greater than 7 units long. 

Hydrolytic enzymes such as lipases and proteases catalyze readily reversible reactions and will often promote reverse hydrolysis at reduced water activities. Water can be removed with desiccants, as an azeotrope with a solvent or through application of a vacuum. Lipases have proven particularly useful in this regard, allowing the formation of esters from alcohols and either free carboxylic acids or esters (see Figure 31.12). 

Cysteine proteases hydrolyze protein amide bonds through formation of a thio-ester bond with an active site cysteine thiol. Cathepsin K is a member of the pa-... 

The overall reaction pathway for the catalytic activity of the thiol proteases is best described by the scheme shown in Figure 12. This mechanism shows the formation of an enzyme-substrate complex which results in the acylation of the enzyme (to form a thiol ester) and its subsequent deacylation, the overall reaction leading to a regeneration of the enzyme, and the elimination of the products of hydrolysis. 

The solubilizing capacity of the choline residue is so pronounced that even substrates combining two hydrophobic amino acids are homogeneously soluble in aqueous buffer without any additional cosolvent. These favorable physical properties were also used in the enzymatic formation of peptide bonds. The amino acid choline ester 38 acts as the carboxyl component in kinetically controlled peptide syntheses with the amino acid amides 39 and 40 [52] (Fig. 11). The fully protected peptides 41 and 42 were built up by means of chymotrypsin in good yields. Other proteases like papain accept choline esters as substrates also, and even butyrylcholine esterase itself is able to generate peptides from these electrophiles. 

Tetrahedral transition state analogues of ester and amide substrates are known to function as efficient enzyme inhibitors of hydrolytic enzymes such as serine and aspartyl proteases as well as metalloproteinases (see Fig. 1.24) [141-144], Although ketals of alkyl or aryl ketones are usually not stable, those of difluroalkyl or trifluoromethyl ketones have considerable stability, as exemplified by their facile formations of the corresponding stable hydrates [145, 146]. Therefore, substrate analogues, containing difluoroalkyl or trifluoromethyl ketone moiety in appropriate positions, have been studied as effective transition state inhibitors of hydrolytic enzymes [141, 147-150],... 

Hydrolases catalyze the addition of water to a substrate by means of a nucleophilic substitution reaction. Hydrolases (hydrolytic enzymes) are the biocatalysts most commonly used in organic synthesis. They have been used to produce intermediates for pharmaceuticals and pesticides, and chiral synthons for asymmetric synthesis. Of particular interest among hydrolases are amidases, proteases, esterases, and lipases. These enzymes catalyze the hydrolysis and formation of ester and amide bonds. 

A very straightforward approach as compared with the existing one (Fig. 3) would have been the direct enzyme-catalyzed peptide formation (cf. Chen et al. [30]) by enantioselective aminolysis of ester 2 with histidine methylester 4 or even racemic histidine ester, as it would resolve the objectives of resolution and coupling in one step. Orientating experiments in which 20 proteases adsorbed on porous glass beads (SIKUG 041/02/120/A, Schott) were in contact with EtOH solutions of 2 and 4 with various water contents, however, did not reveal any reaction. 

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