Parallel 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.

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-... 

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hydrolysis proceeded without involvement of the subtilisin active site Kielbasihski, P. Albrycht, M. Mikolajczyk, M. Unpublished results. 

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 ... 

CI2 (Figure 19.2) is a 64-residue polypeptide inhibitor of serine proteases.23 It has a binding loop (Met-40, which binds in the primary site of chymotrypsin or subtilisin), a single a helix running from residues 12 to 24, and a mixed parallel and antiparallel /3 sheet. The strands and the amphipathic helix interact to form... 

Fig. 5. A representation of the relative dispositions of the side chains of the catalytic aspartates and histidines in (A) RmL, (B) trypsin, (C) subtilisin, and (D) hPL. (A-C) the view is parallel to the plane of the imidazole, looking from where the catalytic serine would be, and along the H bond of His(N81)—Asp(OS2). The histidine nitrogens and the carboxyl oxygens of Asp are identified in A and are the same in B and C. (D) The view is rotated 90° with respect to the preceding view so that the interaction of Asp and His in hPL can be better visualized.

Paralleling the difference in reactivity between primary and secondary hydroxyl groups, the former can be selectively acylated by using PPL in THF [248] or pyridine [47] as the solvent (Scheme 3.18). Unlike most of the other hydrolases, the protease subtilisin is stable enough to remain active even in anhydrous DMF [49]. In general, activated esters such as trihaloethyl esters have been used as acyl donors. 

A great similarity of part of structure of subtilisin and lactate dehydrogenase has been noted (Rao and Rossmaim, 1973). In subtilisin and in carboxypeptidase four parallel central strands of p pleated sheets are super-posable (Rossmann and Argos, 1977). Since these similar structural patterns form structural domains in the corresponding proteins, their evolutionary relationship is discussed in the following paragraphs. 

Parallel approaches have been described for the preparation of polyacrylate-protease conjugates [396-400]. Acryloylation of subtilisin and a-chymotrypsin, followed by mixed polymerization with methyl methacrylate, vinyl acetate, styrene, or ethylvinyl ether, provides insoluble, doped polymethyl methacrylate, polyvinyl acetate, polystyrene, and polyethyl vinyl ether polymers [396]. These biocatalytic plastics perform especially well in hydrophilic and hydrophobic solvents, and have been used for peptide synthesis and the regioselective acylation of sugars and nucleosides. Similarly, modification of subtilisin and thermolysin with PEG monomethacrylate, then copolymerization with methyl methacrylate and trimethylolpropane trimethacrylate furnishes protease-polymethyl methacrylate plastics, which show good activities and stabilities in aqueous, mixed, and low-water and anhydrous organic media [397-400]. The protein-acrylate composites are unique in that they enable catalytic densities as high as 50% w/w. 

---