Three-dimensional structures subtilisin

Figure 11.13 Schematic diagram of the three-dimensional structure of subtilisin viewed down the central parallel p sheet. The N-terminal region that contains the a/p stmcture is blue.

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. 

Drenth, J., et al. Subtilisin novo. The three-dimensional structure and its comparison with subtilisin BPN. 

Comparison of Known Three-dimensional Structures of Subtilisin-type Serine Proteases ... 

Three-dimensional structures of four subtilisin-type enzymes, subtilisin BPN, 36373 subtilisin Carlsberg,37,383 thermitase,39,403 and proteinase K,40,413 are known, but that of aqualysin I has not yet been determined. The Ca atoms of the known structures were superimposed to obtain maximal overlap of the backbone structures, and large parts of all four structures overlap very well (Fig. 12.3) 423 On the basis of such analyses, structurally equivalent core residues (194 residues) are identified, and higher sequence identity was found to correspond to a closer overlap of mainchain atoms in the core (Table 12.1).423... 

As for aqualysin I, its high sequence identity in the core residues with the four proteases (Table 12.1) suggests that the three-dimensional structure of aqualysin I is similar to those of the proteases. The similarity to subtilisins and proteinase K is probably higher than to thermitase. 

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). 

The plot of the stabilities and activities of clones from the first generation S41 random mutant library shows once again that most mutations are detrimental to stability and activity (Fig. 14). However, compared to the esterase library (Fig. 7), there are more mutants with improvements in both properties, suggesting that the two enzymes have different adaptive potentials. This may be due to the relatively poor stability of S41, or it may reflect constraints intrinsic to the three-dimensional structures of the two proteins. Evidence for the former can be found by comparing the results for the first generations of the psychrophilic sub-tilisin S41 and the mesophilic subtilisin E. Screening 864 mutants of S41 yielded nine thermostabilized variants (a hit rate of approximately 1%) (Miyazaki and Arnold, 1999) in contrast, screening 5000 subtilisin E mutants identified five thermostable variants (a hit rate of only 0.1%) (Zhao and Arnold, 1999). 

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. 

Convergence may also occur when the sequence and structure of molecules are very different, but the mechanisms by which they act are similar. Serine proteases have evolved independently in bacteria (e.g. subtilisin) and vertebrates (e.g. trypsin). Despite their very different sequences and three-dimensional structures, in each the same set of three amino acids form the active site. The catalytic triads are His57, Aspl02, and Serl95 (trypsin) and Asp32, His64, and Ser221 (subtilisin) (Doolittle, 1994 A. Tramontano, personal communication). 

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. 

R. Bott, M. Ultsch, A. Kossiakoff, T. Graycar, B. Katz, and S. Power, /. Biol. Chem., 263, 7895 (1988). The Three-Dimensional. Structure of Bacillus amyloliquefadens Subtilisin at 1.8 Angstroms and an Analysis of the Structural Consequences of Peroxide Inactivation. 

As organic solvents are not the natural environment of enzymes, it has been proposed that they may induce changes in the three-dimensional structure of enzymes. The methods developed to study protein structures are difficult to apply to solid enzymes suspended in nonaqueous environments. However, it was possible to show that the structure of subtilisin Carlsberg and y-chymotrypsin is little affected by the passage to organic media (42,43). 

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