Of a subtilisin

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. 

Figure 1. A model of a subtilisin showing the binding cleft and the amino acids of the catalytic triad (Serine-221, Histidine-64, and Aspartate-32)...

Figure 2 Docking of 4-formylphenyl boronic acid into the binding pocket of a subtilisin...

Fig. 12. RMS deviations per residue from the crystal structure during molecular dynamics simulations of (a) subtilisin E at 350°K, (b) subtilisin E at 300°K, (c) 5-3H5 at 350°K, (d) 5-3H5 at 300°K (Colombo and Merz, 1999). Bars indicate the loop region 96-105.

Fig. 5.13. Strategy for the selection of a subtilisin mutant (Submut) displayed on phage [66], Calmodulin (CaM) is also displayed and allows the capture of one product of the reaction via the calmodulin binding peptide (CBD).

Figure 8. Gel filtration on Sephadex G-75 of a subtilisin-digested cellulose from the fungus Penicillium notatum. Void volume, 150 ml. absorbancy at 280 n.m. (O) absorbancy at 570 n.m., ninhydrin reaction (A) cellulase activity...

Figure 14.4.3.2. The dependence of (A) subtilisin Carlsberg and (B) a-chymotrypsin substrate specificity for substrates 1 and 2 on the ratio of their Raoult s law activity coefficients. For the stractures of the substrates 1 and 2, and the solvents a through m in (A) and a to g in (B), refer to Ref 16. [Adapted, by permission, Ifom C.R. Wescott and A.M. Klibanov, Biotechnol. Bioeng., 56, 343(1997)].

Subtilisins are a group of serine proteinases that are produced by different species of bacilli. These enzymes are of considerable commercial interest because they are added to the detergents in washing powder to facilitate removal of proteinaceous stains. Numerous attempts have therefore recently been made to change by protein engineering such properties of the subtilisin molecule as its thermal stability, pH optimum, and specificity. In fact, in 1988 subtilisin mutants were the subject of the first US patent granted for an engineered protein. 

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 /.

Figure 11.16 Substrate-assisted catalysis. Schematic diagram from model building of a substrate, NHa-Phe-Ala-His-Tyr-Gly-COOH (red), bound to the subtilisin mutant His 64-Ala. The diagram illustrates that the His residue of the substrate can occupy roughly the same position in this mutant as His 64 in wild-type subtilisin (see Figure 11.14) and thereby partly restore the catalytic triad.

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 ... 

FIGURE 7.9. The Asn-155- Ala mutation in subtilisin involves deletion of a hydrogen bond between the enzyme and the oxyanion transition state. 

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. 

BERGER, D., ALTMANN T., A subtilisin-like serine protease involved in the regulation of stomatal density and distribution in Arabidopsis thaliana, Gene Dev., 2000, 14, 1119-1131. 

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. 

Novozymes, a subtilisin produced by Bacillus licheniformis, was used by Chen et al ° to carry out a dynamic kinetic resolution of benzyl, butyl, or propyl esters of DL-phenylalanine, tyrosine, and leucine. The hydrolysis was performed at pH 8.5 in 2-methyl-2-propanol/water (19 1) and the freed L-amino acids precipitated. The key feature bringing about continual racemization of the remaining D-amino acid esters was the inclusion of 20 mmol 1 pyridoxal phosphate. 

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