Subtilisin active site

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hydrolysis proceeded without involvement of the subtilisin active site Kielbasihski, P. Albrycht, M. Mikolajczyk, M. Unpublished results. 

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. 

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 molecular weight of these enzymes is around 27,000 g/mol. The active site where the catalysis takes place consists of a catalytic triad of Serine-221, Histidine-64, and Aspartate-32 (the numbers indicates the position of the amino acid in the peptide chain). A model of a subtilisin showing the binding cleft and the amino acids of the catalytic triad is illustrated through Figure 1. 

The closest organic specie to the inorganic boric acid are the boronic acids generally described as R-B(OH)2. Boronic acids have been shown to act as inhibitors of the subtilisins. X-ray crystallographic studies of phenylboronic acid and phenyl-ethyl-boronic acid adducts with Subtilisin Novo have shown that they contain a covalent bond between the oxygen atom of the catalytic serine of the enzyme and the inhibitor boron atom (Matthews et al, 1975 and Lindquist Terry, 1974). The boron atom is co-ordinated tetrahedrally in the enzyme inhibitor complex. It is likely that boric acid itself interacts with the active site of the subtilisins in the same manner. 

Polgar L. and Bender, M.L. (1966) A new enzyme containing a synthetically formed active site. Thiol-subtilisin. J. A/w. Chem. Soc., 88, 3153-3154. 

Figure 3.4 (a) Catalytic efficiencies of subtilisin Carlsberg in THF with different water contents, (b) Polarity of subtilisin s active site (as indicated by the hyperfine splitting constant, A0, of the active-site bound spin label) with different water contents [54]. 

As mentioned earlier (Section 4.2.1.1), empirical rules for the enantioselectivity of hydrolases have been developed. It is important to keep in mind that these rules do not work for all substrates. Most rules are based on pockets, which indicate how the steric bulk of the substituents in the substrate fit into the environment of the active site. Thus, such rules have been suggested for pig liver esterase(PLE) [66], the protease subtilisin [66-68], and certain lipases [69-71]. For secondary alcohols, most lipases follow the simple rule of Kazlauskas, which was developed for Pseudomonas cepacia, and which is depicted in Figure 4.4 [72]. This model implies that the fast-reacting enantiomers binds to the active site as described in Figure 4.4, whereas the slowly reacting one is not able to achieve a comfortable fit, because it will require the large substituent L to fit into the smaller pocket. In contrast to lipases, subtilisin displays opposite enantioselectivity toward secondary alcohols [68]. 

There are formidable problems to be overcome in analyzing mutants of subtilisin that have had their active sites disrupted. Subtilisin is synthesized as a membrane-associated precursor, preprosubtilisin, that is released by autoproteolytic cleavage. 

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