Thermolysin, hydrolysis catalyzed

Scheme 14 Mechanism proposed for peptide hydrolysis catalyzed by thermolysin.

An important difference between thermolysin and carboxypeptidase leads to the major uncertainty in the mechanism of carboxypeptidase. This difference is that the catalytic carboxylate of carboxypeptidase is far more sterically accessible. The crucial question is whether or not the carboxypeptidase-catalyzed hydrolysis of peptides proceeds via general-base catalysis, as in equation 16.26, or via nucleophilic catalysis, as in 16.27. Early kinetic work concentrated on establishing the participation of the various groups in catalysis. 

Thermolysin is a metalloenzyme isolated from Bacillus thermoproteo-lyticus. It is a heat-stable extracellular endopeptidase of molecular weight 34,600. The enzyme catalyzes the hydrolysis of peptide bonds that have the amino group as part of hydrophobic residues such as phenylalanine, isoleucine, or leucine. 

The food industry is a fertile area for biocatalysis applications high-fructose corn syrup (HFCS) from glucose with glucose isomerase, the thermolysin-catalyzed synthesis of the artificial sweetener Aspartame , hydrolysis of lactose for lactose-intolerant consumers, and the synthesis of the nutraceutical i-camitine in a two-enzyme system from "ybutyrobetaine all serve as examples. 

Thus, many metal ions catalyze the hydrolysis of esters [7,8], amides [9], and nitriles [10] via electrophilic activation of the C=0 or C=N group. This type of catalysis is characteristic of coordination complexes and is very common in metalloenzyme-mediated processes. Zinc(II), for example, is a key structural component of more than 300 enzymes, in which its primary function is to act as a Lewis acid (see Chapter 4). The mechanism of action of zinc proteases, e.g., thermolysin, involves electrophilic activation of an amide carbonyl group by coordination to zinc(II) in the active site (Figure 4). 

Enantioselective acylation of amine and hydrolysis of amide are widely studied. These reactions are catalyzed by acylases, amidases and lipases. Some examples are shown in Figure 21.22 Aspartame, artificial sweetener, is synthesized by a protease, thermolysin (Figure 21(a)).22a In this reaction, the L-enantiomer of racemic phenylalanine methyl ester reacted specifically with the a-carboxyl group of N-protected L-aspartate. Both the separation of the enantiomers of the phenylalanine and the protection of the y-carboxyl group of the L-aspartate were unnecessary, which simplified the synthesis. 

Figure 17.22. (a) Thermolysin-catalyzed hydrolysis of peptide analogs showing putative transition state, (b) phosphonamidate peptide analog, and (c) lluoroalkane peptide analog. 

Initial attempts to achieve an enzyme-catalyzed deprotection of the carboxy group of peptides centred around the use of the endopeptidases chymotrypsin, trypsin,and thermolysin.P l Thermolysin is a protease obtained from Bacillus thermoproteolyticus that hydrolyzes peptide bonds on the annino side of the hydrophobic amino acid residues (e.g., leucine, isoleucine, valine, phenylalanine). It cleaved the supporting tripeptide ester H-Leu-Gly-Gly-OEt from a protected undecapeptide (pH 7, rt). The octapeptide, thus obtained, is composed exclusively of hydrophilic annino acids. Due to the broad substrate specificity of thermolysin and the resulting possibility of unspecific peptide hydrolysis, this method is of limited application. 

The first protease-catalyzed reaction in ILs was the Z-aspartame synthesis (Scheme 10.7) from carbobenzoxy-L-aspartate and L-phenylalanine methyl ester catalyzed by thermolysin in [BMIM] [PF ] [ 14]. Subtilisin is a serine protease responsible for the conversion of A -acyl amino acid ester to the corresponding amino acid derivatives. Zhao et al. [90] have used subtilisin in water with 15% [EtPy][CF3COO] as cosolvent to hydrolytically convert a series of A -acyl amino acid esters often with higher enantioselectivity than with organic cosolvent like acetonitrile (Scheme 10.8, Table 10.2). They specifically achieved l-serine and L-4-chlorophenylalanine with an enantiomeric access (ee) of-90% and -35% product yield which was not possible with acetonitrile as a cosolvent [90]. Another example is hydrolysis of A-unprotected amino acid ester in the presence of a cysteine protease known as papain. Liu et al. 

In another research, an approach by using proteases, enzymes that normally hydrolyze peptide bonds in aqueous medium, to perform the reverse reaction (i.e., peptide synthesis or reversed hydrolysis) to produce amphiphilic peptide hydrog-elators that self-assemble to form nanofibrous stmctures was proposed by Ulijin et al. As shown in Fig. 3.34, Fmoc-amino acids (44) and peptides (45) could reacted to form hydrogelator 46 catalyzed by thermolysin (or chymotrypsin), which was able to form transparent gels in water. The result demonstrates that a protease can be used to selectively trigger the self-assembly of peptide hydrogels via reversed hydrolysis [84]. 

Another example where aggregation of a library member drives its synthesis was recently reported by Ulijn ct al. [24, 25]. They used reversible amide bond formation, mediated by thermolysin, which is an enzyme that can catalyze both amide bond hydrolysis and formation, and is only moderately peptide-sequence-dependent. The authors reported that starting from dipeptides and fluorenyl-protected amino acids, the action of thermolysin gives rise to a dynamic mixture of peptides of different lengths (containing typically one to five amino acid residues). When using phenylalanine or leucine as the starting amino acids the... 

As an example of application to a reaction involving a macromolecule, we indicate below the transition state obtained by the combined classical quantum force field in a study of the peptide hydrolysis reaction catalyzed by thermolysin. The subsystem is limited to the substrate, a water molecule, a zinc atom, the side chains of His 143, Glul66, the whole glutamic acid and the histidine 146 moieties (see Figure 10). The transition state represented here corresponds to the proton transfer from Glul43 to the nitrogen atom of the peptidic bond before the breaking of the bond. 

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