Subtilisin, amino acid sequence

The genomic DNA encoding Prl in the other main insect pathogenic Beauveria species, B. brongniartii contains three introns that are 64, 57-, and 61-bp long (Sheng et al., 2006). The deduced amino-acid sequence of the protein shows similarity to that of Prl from M anisopliae (67%), and Prl from B. bassiana (76%). The calculated molecular mass of the Prl precursor is 39121 Da and its pi is 8.0. The protein has three active sites characteristic of subtilisin-like serine proteases. 

Fig. 12.2 Comparison of the amino acid sequence of aqualysin I with those of other subtilisin-type proteases.

Identity of amino acid sequences between subtilisins E and BPN is 86%, so three-dimensional structures of the two enzymes are considered to be very similar. In the case of subtilisin BPN, residues 61 and 98 are located on the loop and turn structure, respectively, both of which connect /3-strand and a-helix (Fig. 12.5). Solvent exposures of the residues are both 9,45) indicating their presence on the surface of the enzyme molecule. The distance between the a-carbons of the two residues is 5.8 A. Accordingly, the positions seem appropriate for cysteine residues to form a disulfide bond without any strain in the enzyme structure. The disulfide bond formed is located close to the active site so as to stabilize the wall of the active-site pocket (Fig. 12.5). 

To establish the amino acid sequence unequivocally it is necessary to have peptides with overlapping sequences. This may be accomplished by determining the sequence of fragments obtained from treating a second aliquot of the protein with chymotrypsin. If these fragments are then treated with trypsin as a check, peptides identical to those obtained previously by successive treatment with trypsin and chymotrypsin are obtained. Other proteolytic enzymes, such as pepsin, subtilisin, and papain, with wider specificity than trypsin and chymotrypsin have proved useful in sequencing of some proteins. 

Residues of serine, histidine, and aspartic acid will be designated Ser-195, His-57, and Asp-102 in the following discussion. The numbers refer to their location in the amino acid sequence of chymotrypsinogen (22), but they have no absolute significance for other enzymes and would occur in a different order if they had been based on the subtilisin sequence. 

The crystal structure of subtilisin BPN dispelled this uncertainty. As already mentioned, the subtilisins and the pancreatic enzymes are dissimilar in amino acid sequence, and they proved to be dissimilar in their gross three-dimensional structure. However, the components of their catalytic site do not differ. Both enzyme groups have the same catalytic triad with hydrogen bonds linking serine to N-3 of histidine and N-1 of histidine to a buried side chain of aspartic acid (29). Since the two enzyme groups are products of different evolutionary pathways, it follows almost inescapably that this striking homology is dictated by necessity and that the buried aspartic acid is essential for catalysis. 

Chymotrypsin and subtilisin also differ in their amino acid sequences, number of disulfide bridges (chymotrypsin has five, whereas subtilisin has none), and overall three-dimensional structures. The striking difference in structure and common catalytic mechanism are taken as evidence of an independent but convergent evolutionary process. 

The first crystallization of cytochrome 65, obtained from pig liver was reported by Raw and Coli in 1959 (10 ). Kajihara and Hagihara (129) obtained three crystalline cytochrome be preparations from rabbit liver microsomes, two from trypsin extracts, and one from Nagarse (subtilisin BPN ) extracts. The three preparations crystallized in entirely different shapes. Calf liver cytochrome bs has also been crystallized by Mathews and Strittmatter (ISS). The preparations by the latter two groups have been used for studies on the amino acid sequence and the three-dimensional structure, respectively. 

CPA seems to occur only in mammals, but it should be noted that there is a related Zn endopeptidase, ther-molysin (EC 3.4.24.4), in thermophilic bacterium Bacillus thermoproteolyticus. Although its amino acid sequence and three-dimensional structure are unrelated to CPA. the active site structure is similar, and the mechanism of action also seems to be similar.This is an example of convergent evolution just like the case of serine proteases mammalian chymotrypsin and microbial subtilisin. 

Figure 25.3 shows the relationship of active site of serine hydrolases. The serine hydrolases include serine proteases, lipases, and PHB depolymerases. A common feature of the serine proteases is the presence of a specific amino acid sequence -Gly-Xl-Ser-X2-Gly-. The catalytic mechanism of these enzymes is very similar and the catalytic center consists of a triad of serine, histidine, and aspartate residues [54]. The serine from this sequence attacks the ester bond nucleophilically [55]. Lipases and PHB depolymerases also have a common amino acid sequence around the active site, -Gly-Xl-Ser-X2-Gly-. These serine hydrolases may share a similar mechanism of substrate hydrolysis [21, 56]. In terms of origin of enzymes, it would be wise to consider that the enzyme had wide substrate specificity initially, and then it started to evolve gradually for each specific substrate. In the case of polyester hydrolysis, lipases showed the widest substrate specificity among serine hydrolases for hydrolysis of various polyesters ranging from a-ester bonds to (o-ester bonds. PHB depolymerases would become more specific for microbial PHB that has / -ester bonds, though it could also hydrolyze other polyesters that have -ester and y-ester bonds. Serine proteases such as proteinase K, subtilisin, a-chymotrypsin, elastase, and trypsin hydrolyze only optically active PLLA with a-ester bonds and various proteins with a-amido bonds. 

Fig. 1. Alignment of tryptic, chymotryptic and cyanogen bromide fragments and of peptides derived from them with the amino acid sequence of thermitase. The regions sequenced are marked by a full line under the horizontal bars representing the individual peptides. Individual symbols T - tryptic peptide, C - chymotryptic peptide, S -subtilisin peptide, CB - cyanogen bromide fragment.

Odani, S., Koide, T., Ono, T. (1983). The complete amino acid sequence of barley trypsin inhibitor. /, of Biological Chemistry, Vol. 258, No. 13, pp. 7998-8003, ISSN 0021-9258 Ohtsubo, K, Richardson, V. (1992). The amino acid sequence of a 20 kDa bifunctional subtilisin alpha-amylase inhibitor from brain of rice (Oryza sativa L.) seeds. FEBS, Vol. 309, pp. 68-72, ISSN 1742-4658... 

Comparison (or alignment) of amino acid sequences, also called homology search, often provides first-hand information on such conserved structural features and enables one to classify enzymes into families and predict the possible function of a new enzyme (86). A family of enzymes usually folds into similar 3-D structures, at least at the active site region. A typical example is the serine protease family whose members—trypsin, chymotrypsin, elastase, and subtilisin—commonly contain three active-site residues, Asp/His/Ser, which are known as the catalytic triad or charge relay system. Another example is the conserved features of catalytic domains of the highly diverse protein kinase family. In this kinase family, the ATP-binding (or phosphate-anchoring) sites present a consensus sequence motif of Gly-X-Gly-X-X-Gly (67,87). 

Despite their lack of stabilizing disulfide bridges Potl inhibitors feature a common, stable fold. The N-terminus is coiled, although in some structures a small /3-strand has been identified. After a turn the structure adopts an a-helical structure, followed by a turn and an other /3-strand. The sequence then features an extended turn or loop motif that contains the reactive site of the inhibitor before it proceeds with a /3-strand running almost parallel to the /3-strand after the a-helix. After another turn and coiled motif a short /3-strand antiparallel to the other /3-strands precedes the coiled C-terminus. Usually the N-terminal residue in the reactive site is an acidic residue followed by an aromatic amino acid, that is, tyrosine or phenylalanine. Figure 11 shows the complex of chymotrypsin inhibitor (Cl) 2 with subtilisin, the hexamer of Cl 2 from H. vulgare and a structural comparison with a trypsin inhibitor from Linum usitatissimum ... 

The enzyme consists of a single polypeptide chain of Mr 13 680 and 124 amino acid residues.187,188 The bond between Ala-20 and Ser-21 may be cleaved by subtilisin. Interestingly, the peptide remains attached to the rest of the protein by noncovalent bonds. The modified protein, called ribonuclease S, and the native protein, now termed ribonuclease A, have identical catalytic activities. Because of its small size, its availability, and its ruggedness, ribonuclease is very amenable to physical and chemical study. It was the first enzyme to be sequenced.187 The crystal structures of both forms of the enzyme were solved at 2.0-A resolution several years ago.189,190 Subsequently, crystal structures of many complexes of the enzyme with substrate and transition analogues and products have been solved at very high resolution.191 Further, because the catalytic activity depends on the ionizations of two histidine residues, the enzyme has been extensively studied by NMR (the imidazole rings of histidines are easily studied by this method—see Chapter 5). 

Very recently it was shown that the X-ray analysis of eglin c and its complex with subtilisin revealed a similar type of global structural changes induced by the subtilisin binding.29 Obviously SSI has no sequence or structural similarity with eglin c, which is a monomeric protein having 70 amino acid residues produced by the leech Hirudo medicinalis. It will thus be very interesting to compare the structural as well as biochemical characteristics of these two inhibitors. 

The sequence of aqualysin Il6) (AQU) is shown compared with those of proteinase Kl7) (PRO), thermitase22 (THE), subtilisin BPN 18 (BPN), and subtilisin El9) (E). Identical amino acids with those of aqualysin I are shown by hyphen (—). Open space is the position where a corresponding amino acid is absent. The numbering above the sequences refers to aqualysin I, and that below the sequences to subtilisins. Asterisks indicate the active-site residues, Asp, His, and Ser. 

---