Design and Synthesis of a Peptide Having Chymotrypsin-Like Esterase Activity

Abstract

A peptide having enzyme-like catalytic activity has been designed and synthesized. Computer modeling was used to design a bundle of four short parallel amphipathic helical peptides bearing the serine protease catalytic site residues serine, histidine, and aspartic acid at the amino end of the bundle in the same spatial arrangement as in chymotrypsin (ChTr). The necessary "oxyanion hole" and substrate binding pocket for acetyltyrosine ethyl ester, a classical ChTr substrate, were included in the design. The four chains were linked covalently at their carboxyl ends. The peptide has affinity for ChTr ester substrates similar to that of ChTr and hydrolyzes them at rates approximately 0.01 that of ChTr; total turnovers greater than 100 have been observed. The peptide is inhibited by ChTr specific inhibitors and is inactive toward benzoyl arginine ethyl ester, a trypsin substrate. The peptide is inactivated by heating above 60 degrees C, but recovers full catalytic activity upon cooling and lyophilization from acetic acid.

α-Helical Protein Assembly Motifs

“…The simplicity of the four-helix bundle motif has made it ideal as a template for several attempts at de novo protein design (52)(53)(54). An interesting variation of this de novo design concept is also offered by the template-assembled synthetic protein approach described by Mutter's group (55).…”

A differential scanning calorimetric study of the thermal unfolding of apo‐ and holo‐cytochrome b562

“…The fourhelix bundle is a very common structural motif, and the relative simplicity of the structure makes it an important target for de novo design efforts (DeGrado et al, 1989;Hahn et al, 1990;Hecht et al, 1990;Robertson et al, 1994;Bryson et al, 1995;Kalsbeck et al, 1996). Additionally, comparing apo-and holoprotein pairs provides valuable information regarding the importance of prosthetic groups to structure, stability, and assembly (Cocco & Lecomte, 1990;Moore et al, 1991;Feng et al, 1994).…”

Synthesis of Functionalised Nucleosides for Incorporation into Nucleic Acid-Based Serine Protease Mimics

“…The hydrolysis is realized by the unique collaboration between Histidine-57 (His-57), and Aspartate-102 (Asp-102) [5]. Intrigued by this performance, several groups have tried to construct model systems with hydrolytic activity and different peptide based systems have been developed in this context [6][7][8][9][10][11][12][13][14][15][16]. We must bear in mind that stable secondary structures such as helices only become important for rather long sequences of polypeptides.…”