Subtilisin structure

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. 

Using X-ray structure data, amino acids which were believed to affect the desired characteristics were identified. Using either site-directed mutagenesis or "cassette" mutagenesis (8) different amino acid substitutions were made in the subtilisin structural gene (2,9). 

Stahl, M.L. and Ferrari, E. (1984) Replacement of the Bacillus subtilis subtilisin structural gene with an in vitro-derived deletion mutation. 

Bode, W., Papamokos, E., Musil, D. The high-resolution X-ray crystal structure of the complex formed between subtilisin Carlsberg and eglin c, an elastase inhibitor from the leech Hirudo medicinalis. Eur. J. Biochem. 166 (1987) 673-692... 

McPhalen, C. A., James, M. N. G. Structural comparison of two serine proteinase-protein inhibitor complexes Eglin-C-Subtilisin Carlsberg and CI-2-subtilisin novo. Biochemistry 27 (1988) 6582-6598... 

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.

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. 

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

A structural anomaly in subtilisin has functional consequences Transition-state stabilization in subtilisin is dissected by protein engineering Catalysis occurs without a catalytic triad Substrate molecules provide catalytic groups in substrate-assisted catalysis Conclusion Selected readings... 

Connections between /3-strands are of two types—hairpins and cross-overs. Hairpins, as shown in Figure 6.27, connect adjacent antiparallel /3-strands. Cross-overs are necessary to connect adjacent (or nearly adjacent) parallel /3-strands. Nearly all cross-over structures are right-handed. Only in subtilisin and phosphoglucoisomerase have isolated left-handed cross-overs been identi-... 

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... 

SCF, see Self-consistent field treatment (SCF) Schroedinger equation, 2,4,74 Secular equations, 6,10, 52 solution by matrix diagonalization, 11 computer program for, 31-33 Self-consistent field treatment (SCF), of molecular orbitals, 28 Serine, structure of, 110 Serine proteases, 170-188. See also Subtilisin Trypsin enzyme family comparison of mechanisms for, 182-184, 183... 

Thacker, C., Peters, K., Srayko, M. and Rose, AM. (1995) The bli-4 locus of Caenorhabditis elegans encodes structurally distinct kex2/subtilisin-like endoproteases essential for early development and adult morphology. Genes and Development 9, 956—971. 

---