Enzymes thermolysin

The improvement of enzyme like MIP is currently another area of intense research. Beside the use of the MIP themselves as catalysts, they may also be applied as enhancer of product yield in bio-transformation processes. In an exemplary condensation of Z-L-aspartic acid with L-phenylalanine methyl ester to Z-aspartame, the enzyme thermolysin was used as catalyst. In order to shift the equilibrium towards product formation, a product imprinted MIP was added. By adsorbing specifically the freshly generated product from the reaction mixture, the MIP helped to increase product formation by 40% [130]. MIP can also be used to support a physical process. Copolymers of 6-methacrylamidohexanoic acid and DVB generated in the presence of calcite were investigated with respect to promotion of the nucleation of calcite. Figure 19 (left) shows the polymer surface with imprints from the calcite crystals. When employing these polymers in an aqueous solution of Ca2+ and CO2 the enhanced formation of rhombohedral calcite crystals was observed see Fig. 19 (right) [131]. 

There also are numerous enzymes that use bound metal ions to form complexes with substrates. In these enzymes, the metal ion usually serves as an electrophilic functional group rather than as a nucleophile. Carbonic anhy-drase, for example, contains a Zn+2 ion that binds one of the substrates, hydroxide ion, as a ligand. The bound OH reacts with the other substrate, C02. In alcohol dehydrogenase, and in the proteolytic enzymes thermolysin and car-boxypeptidase A, a Zn+2 ion forms a complex with a carbonyl oxygen atom of the substrate. The withdrawal of electrons by the Zn+2 increases the partial positive charge on the carbonyl carbon and thus promotes reaction with a nucleophile. 

Figure 10-3. The environment of the four structural calcium ions in the enzyme thermolysin isolated from Bacillus thermoproteolyticus. All of the calcium ions are associated with oxygen donor ligands.

Finally, enzymes that bind metal cofactors such as Zn + and Mg + can use their properties as Lewis acids, for example, electron pair acceptors. An example is the enzyme thermolysin, whose mechanism is illustrated in Fig. 9. In this enzyme, glutamate-143 acts as an active site base to deprotonate water for attack on the amide carbonyl, which is at the same time polarized by coordination by an active site Zn + ion (6). The protonated glutamic acid then probably acts as an acidic group for the protonation of the departing amine. 

The metalloproteases constitute the final major class of peptide-cleaving enzymes. The active site of such a protein contains a bound metal ion, almost always zinc, that activates a water molecule to act as a nucleophile to attack the peptide carbonyl group. The bacterial enzyme thermolysin and the digestive enzyme carboxypeptidase A are classic examples of the zinc proteases. Thermolysin, but not carboxypeptidase A, is a member of a large and diverse family of homologous zinc proteases that includes the matrix metalloproteases, enzymes that catalyze the reactions in tissue remodeling and degradation. 

Figure 23-1 illustrates the application of equation 23-4 in the determination of the hydrolysis of a substrate by the enzyme thermolysin. The parameters... 

Metalloprotease inhibitors - also known as metalloproteases or zinc proteases - are proteolytic enzymes of which the activity depends on metal ions, normally bound Zn -. Examples of metalloproteases are the pancreatic enzymes carboxypeptidase A and B, elastase, the well-characterized bacterial enzyme thermolysin and the collagenase family (found in both bacterial and mammalian cells, fibroblast collagenase, neutrophil elastase, gelatinase). 

Another example of a protease-catalyzed commercial process, which in this case uses the enzyme in a synthetic mode, is the completely regio- and stereoselective production of the low-caloric sweetener aspartame developed by DSM-TOSOH [15] (Fig. 7.9). Aspartame is a dipeptide consisting of the amino acids phenylalanine and aspartic acid, which are coupled by the enzyme thermolysin from Bacillus thermoproteolyticus. For an efficient coupling, relatively high temperatures are required and the amount of water in the system must be kept low to drive the reaction in the desired direction. Thermolysin, which is a metallo-endoprotease, meets these two requirements. It is thermostable, and it works in an organic solvent, which is required to keep the water activity low. In practice, however, organic solvents were not necessary, since the product aspartame forms an insoluble complex with unreacted D-Phe-OMe, which crystallizes out of the aqueous medium. 

For the mechanistically related enzymes thermolysin and stromdysin, a strong case has been made supporting the hypothesis that the amine of leaving groups during turnover of peptide substrates is fully protonated in the transition state and thus requires no assistance from a general acid [71]. 

Matthews et al. have determined the structure of the extracellular proteolytic enzyme, thermolysin, to 2.3 A resolution. This enzyme of molecular weight 37 500 contains one zinc and four calcium ions, and is interesting because of its imusual heat stability. The active site contains a zinc atom tetrahedrally co-ordinated to His-142, His-146, Glu-166, and a water molecule, and in this way resembles carboxypeptidase A. The precise details of the co-ordination of the calcium ions are not reported. However, it is of interest that two have a centre-to-centre distance of 3.8 A and are located within an interior region of the protein, surrounded by carbonyl and carboxylate groups. Loss of calcium does not hinder proteolysis at room temperature, but the enzyme is no longer heat stable. Preliminary X-ray data are also reported for the acidic protease from the fungus Rhizopus chinensis. ... 

But profit is not the goal, nor should it be the product, of academic research. Our products are ideas and well-trained students, who can go to industry and solve relevant problems. If we really want to undmtand how a protein binds a small molecule, and bow to design the best inhibitor of an enzyme, thermolysin is just as useful as the angiotensin-converting enzyme. 

SLBO is sometimes called a quanto-classical atom. This modelling tool has demonstrated its efficiency in several applications such as the study of a peptide hydrolysis by an enzyme (Thermolysin) [40]... 

After reaction with the labeled reagents, the protein is treated with proteolytic enzymes thermolysin, chymotrypsin, and trypsin have been used for proteolysis of the labeled macromolecule. The resulting peptides are separated by high-pressure liquid chromatography (HPLC), and the amount of radioactivity of each eluted peak is measured. Then the labeled peptides are analyzed after acid hydrolysis and the amount of radioactive label incorporated into individual amino acid side chains is measured. 

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