Thermolysin, reaction

Overall the mechanism of the thermolysin reaction including the role of the Zn2 + in catalysis is given in Fig. 5. This proposed mechanism is based on inhibitor data and x-ray structure determination. 

An analysis of phosphonamidate and phosphonate transition-state analogs for the thermolysin reaction was reported (95, 96) and the data were interpreted to suggest that one hydrogen bond could contribute 4 kcal/mol binding to stabilize the formation of the tetrahedral transition state. A reevaluation was re-... 

Although equihbrium-controUed peptide synthesis has been successfully used on a number of occasions, including thermolysin-catalyzed synthesis of aspartame (126) and semisynthesis of insulin (127), the method has a significant drawback a water-miscible organic cosolvent added to the reaction medium to suppress the ionization of unactivated carboxy components significantly reduces the reaction rate. 

In the first publication describing the preparative use of an enzymatic reaction in ionic liquids, Erbeldinger et al. reported the use of the protease thermolysin for the synthesis of the dipeptide Z-aspartame (Entry 6) [34]. The reaction rates were comparable to those found in conventional organic solvents such as ethyl acetate. Additionally, the enzyme stability was increased in the ionic liquid. The ionic liquid was recycled several times after the removal of non-converted substrates by extraction with water and product precipitation. Recycling of the enzyme has not been reported. It should be noted, however, that according to the log P concept described in the previous section, ethyl acetate - with a value of 0.68 - may interfere with the pro-... 

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

Tetrhedral intermediate, 172 Thermodynamic cycles, 186 Thermolysin, zinc as cofactor for, 204 Thrombin, 170 Torsional potential, 111 Transition states, 41-42,44, 45,46, 88, 90-92 in amide hydrolysis, 219-221 oxyanion hole and, 181 stabilization of, 181,181 carbonium ion, 154,155,156-161, 167-169 for gas-phase reactions, 43... 

The observed normal isotope effect of 1.9 provides further evidence supporting the role of Asp55 as the general base. Namely, a normal isotope effect of 1.9 is most consistent with general base catalysis by an amino acid side chain, as inverse isotope effects are commonly observed when a zinc-bound water molecule, or hydroxide, is the attacking nucleophile. For example, the zinc-containing enzymes AMP deaminase [111], thermolysin [112], stromelysin [113], and a desuccinylase [114] are each believed to utilize a zinc-bound water as the nucleophile, and all of these reactions are characterized by an inverse deuterium isotope effect. This inverse isotope effect is thought to result from a dominant... 

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]. 

Electron paramagnetic resonance (continued) cobalt-thermolysin complex, 28 334, 335 exchange reactions, 31 106-107 glutamine synthetase, 28 358-364 invisible oxygen species, 31 94-95 metalloenzymes, 28 324, 326 metal particle size distribution, 36 99-100, 104... 

Bartlett etal. [14] described the synthesis of a series of fluoroolefin tripeptide isosteres Cbz-Glyi/r[(Z)-CF=CH]LeuXaa (Xaa = Gly, Ala, Leu, Phe, and NH2) (10) (Scheme 3) as the ground-state analog inhibitors of thermolysin. Treatment of acid (15) with ammonia gave 10e. The inhibitors 10a-d are formed by conventional amide acid coupling reactions of suitably protected amino acids followed by saponification with lithium hydroxide. 

The metalloprotease thermolysin, obtained from Bacillus thermoproteolyticus, a strain of B. stearothermophilus, is used as a crude preparation in an aqueous medium. The enzyme is recovered from the reaction mixture by ultrafiltration with a yield of >95%. 

Isowa, Y., Ohmori, M., Ichikawa, T., Mori, K., Nonaka, Y. andKihara, K. (1979) The thermolysin-catalyzed condensation reactions of N-substituted aspartic and glutamic acids with phenylalanine alkyl esters. Tetrahedron Lett., 28, 2611-2612. 

FIGURE 17. Favored reaction mechanism of thermolysin. Adapted with permission from References 9 and 140. Copyright (2003) and (1998) ACS... 

Figure 3.6 Initial rates of the reaction between Z-Gly-Gly-Phe and Phe-NH2 catalyzed by thermolysin in ferf-amyl alcohol containing various cosolvents [31].

Proteases have been much less studied than lipases in ionic liquid media and generally require the presence of water for activity. We note that the thermolysin-catalyzed amide coupling of benzoxycarbonyl-L-aspartate and L-phenylalanine methyl ester into Z-aspartame in [BMIm][PF6] was an early example of an enzymatic reaction in an ionic liquid medium [8]. 

There are a number of reported solid-to-solid reactions where products precipitated as salts rather than as neutral compounds. The thermolysin-catalyzed production of the potassium salt of Z-aspartame [51, 52], the commercialized process of aspartame synthesis, where a salt of cationic D-Phe-OMe and anionic Z-aspartame precipitates [53], and the enzymatic conversion of solid Ca-maleate to solid Ca-D-malate [44] are examples of such behavior. 

In the thermolysin-catalyzed synthesis of the potassium salt of Z-aspartame (Scheme 12.1), the reaction rate was found to be strongly dependent on the amount of basic salt (KHC03, Figure 12.3) added to the system, as mentioned above [51]. 

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. 

K. Oyama, K. Kihara, and Y. Nonaka, On the mechanism of the action of thermolysin kinetic study of the thermolysin-catalyzed condensation reaction of N-benzyloxy-carbonyl-t-aspartic add with L-phenyl-alanine methyl ester, J. Chem. Soc. Perkin Trans. 2 1981a, 356-360. 

Plots of initial reaction rates as a function of percentage active subtilisin in the biocatalyst were found to be linear for all biocatalyst preparations. Thus, enzyme activation, as high as 3750-fold in hexane for the transesterification of N-Ac-i-Phe-OEt with n-PrOH, is a manifestation of intrinsic enzyme activation and not of relaxation of diffusional limitations resulting from diluted enzyme preparations. Thus, the above-mentioned hypothesis was refuted. As similar results were found for the metalloprotease thermolysin, activation due to lyophilization in the presence of KC1 may be a general phenomenon. 

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