Subtilisin activity

Our parallel experiments, in which subtilisin Carlsberg was used to promote hydrolysis of A-acetyl-A-benzyl arenesulfinamides, led to exclusive S-N bond breaking. However, the recovered substrates were racemic. Moreover, blank experiments showed that a spontaneous chemical hydrolysis contributed to the process to a much higher degree than that in the cases shown in Ref. 47. Hence, a conclusion was drawn that in our case the hydrolysis proceeded without involvement of the subtilisin active site Kielbasihski, P. Albrycht, M. Mikolajczyk, M. Unpublished results. 

Figure 3. pH profiles of wild type and two variant subtilisins. Activity was assayed with synthetic substrates as described (2) at the indicated pH. 

RichJO and DordickJS. Controlling Subtilisin Activity and Selectivity in Organic Media by Imprinting with Nucleophilic Substrates. /Am Chem Soc 1997 119 3245-3252. 

Engineering the pH Proji/e of Subtilisin. The activity of subtilisin BPN increases between pH 6 and 8 as His64 7.2) is deprotonated (68). Changes in... 

Enzymatic acylation reactions offer considerable promise in the synthesis of specific ester derivatives of sucrose. For example, reaction of sucrose with an activated alkyl ester in /V, /V- dim ethyl form am i de in the presence of subtilisin gave 1 -0-butyrylsucrose, which on further treatment with an activated fatty acid ester in acetone in the presence of Hpase C. viscosum produced the 1, 6-diester derivative (71,72). 

Subtilisin (from Bacillus subtilis) [9014-01-1 ] [EC 3.4.21.62]. Purified by affinity chromatography using 4-(4-aminophenylazo)phenylarsonic acid complex to activated CH-Sepharose 4B. [Chandraskaren and Dhai Anal Biochem 150 141 7955]. 

The active site of subtilisin is outside the carboxy ends of the central p strands analogous to the position of the binding sites in other a/p proteins as discussed in Chapter 4. Details of this active site are surprisingly similar to those of chymotrypsin, in spite of the completely different folds of the two enzymes (Figures 11.14 and 11.9). A catalytic triad is present that comprises residues Asp 32, His 64 and the reactive Ser 221. The negatively charged oxygen atom of the tetrahedral transition state binds in an oxyanion hole,... 

Figure 11.14 Schematic diagram of the active site of subtilisin. A region (residues 42-45) of a bound polypeptide inhibitor, eglin, is shown in red. The four essential features of the active site— the catalytic triad, the oxyanion hole, the specificity pocket, and the region for nonspecific binding of substrate—are highlighted in yellow. Important hydrogen bonds between enzyme and inhibitor are striped. This figure should be compared to Figure 11.9, which shows the same features for chymotrypsin. (Adapted from W. Bode et al., EMBO /.

All the four essential features of the active site of chymotrypsin are thus also present in subtilisin. Furthermore, these features are spatially arranged in the same way in the two enzymes, even though different framework structures bring different loop regions into position in the active site. This is a classical example of convergent evolution at the molecular level. 

Serine proteinases such as chymotrypsin and subtilisin catalyze the cleavage of peptide bonds. Four features essential for catalysis are present in the three-dimensional structures of all serine proteinases a catalytic triad, an oxyanion binding site, a substrate specificity pocket, and a nonspecific binding site for polypeptide substrates. These four features, in a very similar arrangement, are present in both chymotrypsin and subtilisin even though they are achieved in the two enzymes in completely different ways by quite different three-dimensional structures. Chymotrypsin is built up from two p-barrel domains, whereas the subtilisin structure is of the a/p type. These two enzymes provide an example of convergent evolution where completely different loop regions, attached to different framework structures, form similar active sites. 

Husum et al. found that the hydrolytic activities of P-galactosidase from E. coli and the protease subtilisin in a 50 % aqueous solution of the water-miscible ionic liquid [BMIM][Bp4] were comparable to those in 50 % aqueous solutions of ethanol or acetonitrile (Entry 9) [37]. 

The serine proteases are the most extensively studied class of enzymes. These enzymes are characterized by the presence of a unique serine amino acid. Two major evolutionary families are presented in this class. The bacterial protease subtilisin and the trypsin family, which includes the enzymes trypsin, chymotrypsin, elastase as well as thrombin, plasmin, and others involved in a diverse range of cellular functions including digestion, blood clotting, hormone production, and complement activation. The trypsin family catalyzes the reaction ... 

The elucidation of the X-ray structure of chymotrypsin (Ref. 1) and in a later stage of subtilisin (Ref. 2) revealed an active site with three crucial groups (Fig. 7.1)-the active serine, a neighboring histidine, and a buried aspartic acid. These three residues are frequently called the catalytic triad, and are designated here as Aspc Hisc Serc (where c indicates a catalytic residue). The identification of the location of the active-site groups and intense biochemical studies led to several mechanistic proposals for the action of serine proteases (see, for example, Refs. 1 and 2). However, it appears that without some way of translating the structural information to reaction-potential surfaces it is hard to discriminate between different alternative mechanisms. Thus it is instructive to use the procedure introduced in previous chapters and to examine the feasibility of different... 

FIGURE 9.5. The potential surface for the 0"C = 0— 0-C-0" step in amide hydrolysis in solution, where the surface is given in terms of the angle 0 and the distance b. The heavy contour lines are spaced by fi (at room temperature) and can be used conveniently in estimating entropic effects. The figure also shows the regions (cross hatched) where the potential is less than for the corresponding reaction in the active site of subtilisin. 

Enzyme active sites, 136,148, 225. See also Protein active sites in carbonic anhydrase, 197-199 in chymotrypsin, 173 in lysozyme, 153, 157 nonpolar (hypothetical site), 211-214 SNase, 189-190,190 steric forces in, 155-158, 209-211, 225 in subtilisin, 173 viewed as super solvents, 227 Enzyme cofactors calcium ... 

Subtilisin, 170 active site of, 171,173 autocorrelation function of, 216, 216 potential surfaces for, 218 site-specific mutations, 184, 185, 187-188 Sugars, see Oligosaccharides Surface-constrained solvent model, 125... 

The second group of studies tries to explain the solvent effects on enantioselectivity by means of the contribution of substrate solvation to the energetics of the reaction [38], For instance, a theoretical model based on the thermodynamics of substrate solvation was developed [39]. However, this model, based on the determination of the desolvated portion of the substrate transition state by molecular modeling and on the calculation of the activity coefficient by UNIFAC, gave contradictory results. In fact, it was successful in predicting solvent effects on the enantio- and prochiral selectivity of y-chymotrypsin with racemic 3-hydroxy-2-phenylpropionate and 2-substituted 1,3-propanediols [39], whereas it failed in the case of subtilisin and racemic sec-phenetyl alcohol and traws-sobrerol [40]. That substrate solvation by the solvent can contribute to enzyme enantioselectivity was also claimed in the case of subtilisin-catalyzed resolution of secondary alcohols [41]. 

The lipase-catalyzed DKRs provide only (/ )-products to obtain (5 )-products, we needed a complementary (5 )-stereoselective enzyme. A survey of (5 )-selective enzymes compatible to use in DKR at room temperature revealed that subtilisin is a worthy candidate, but its commercial form was not applicable to DKR due to its low enzyme activity and instability. However, we succeeded in enhancing its activity by treating it with a surfactant before use. At room temperature DKR with subtilisin and ruthenium catalyst 5, trifluoroethyl butanoate was employed as an acylating agent and the (5 )-products were obtained in good yields and high optical purities (Table 3)P... 

Serine proteases usually show primary specificity (occupation of subsite Si) for positively charged arginine or lysine (trypsin, plasmin, plasminogen activators, thrombin), large hydrophobic side chains of phenylalanine, tyrosine, and tryptophan (chymotrypsin, cathepsin G, chymase, and subtilisin), or small aliphatic side chains (elastases). Nevertheless, there are a large number of variations and in many cases, other subsites like S2 and S3 are more discriminating while maintaining the... 

DNA polymerase I has been purified to homogeneity. When the pure enzyme is treated with subtilisin, a proteolytic enzyme from Bacillus subtilis, the polymerase is cleaved into two pieces. The small fragment retains the 5 to 3 nuclease activity, whereas the larger piece, called a Klenow fragment, has both polymerase activity and the 3 to 5 exonuclease activity. The Klenow fragment is sold commercially for use in labeling DNA for use in detecting recombinant DNA. 

Fontes tt al. [224,225 addressed the acid—base effects of the zeolites on enzymes in nonaqueous media by looking at how these materials affected the catalytic activity of cross-linked subtilisin microcrystals in supercritical fluids (C02, ethane) and in polar and nonpolar organic solvents (acetonitrile, hexane) at controlled water activity (aw). They were interested in how immobilization of subtilisin on zeolite could affected its ionization state and hence their catalytic performances. Transesterification activity of substilisin supported on NaA zeolite is improved up to 10-fold and 100-fold when performed under low aw values in supercritical-C02 and supercritical-ethane respectively. The increase is also observed when increasing the amount of zeolite due not only to a dehydrating effect but also to a cation exchange process between the surface proton of the enzyme and the sodium ions of the zeolite. The resulting basic form of the enzyme enhances the catalytic activity. In organic solvent the activity was even more enhanced than in sc-hexane, 10-fold and 20-fold for acetonitrile and hexane, respectively, probably due to a difference in the solubility of the acid byproduct. 

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