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Design of Peptide Enzymes (Pepzymes): Surface-Simulation Synthetic Peptides that Mimic the Chymotrypsin and Trypsin Active Sites Exhibit the Activity and Specificity of the Respective Enzyme

M. Zouhair Atassi and Taghi Manshouri
Proceedings of the National Academy of Sciences of the United States of America
Vol. 90, No. 17 (Sep. 1, 1993), pp. 8282-8286
Stable URL: http://www.jstor.org/stable/2363010
Page Count: 5
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Design of Peptide Enzymes (Pepzymes): Surface-Simulation Synthetic Peptides that Mimic the Chymotrypsin and Trypsin Active Sites Exhibit the Activity and Specificity of the Respective Enzyme
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Abstract

Two 29-residue peptides were prepared, one of which (ChPepz) was designed by surface-simulation synthesis to mimic the active site of α-chymotrypsin, and the other (TrPepz), which contained four substitutions relative to ChPepz, was fashioned after the active site of trypsin. Each peptide was cyclized by a disulfide bond. The ChPepz monomer effected hydrolysis of the ester group in N-benzoyl-L-tyrosine ethyl ester, an α-chymotrypsin substrate, with Km and kcat values that were comparable to those of α-chymotrypsin. ChPepz was completely inactivated by diisopropyl fluorophosphate (DIFP), L-1-p-tosylamino-2-phenylethyl chloromethyl ketone (TPCK), or reduction of the disulfide bond. It had no catalytic activity on N-tosyl-L-arginine methyl ester, a trypsin substrate. On the other hand, TrPepz, which had no effect on N-benzoyl-L-tyrosine ethyl ester, hydrolyzed N-tosyl-L-arginine methyl ester with a Km value that was essentially identical to that of trypsin, but its kcat value was almost half that of trypsin. TrPepz was fully inactivated by reduction of the disulfide bond, by DIFP, or by phenylmethylsulfonyl fluoride but not by TPCK. It was also completely inhibited by soybean trypsin inhibitor, bovine pancreatic trypsin inhibitor, and human α1-antitrypsin. ChPepz and TrPepz hydrolyzed proteins (myoglobin and casein) to give panels of peptides that were similar to those of the same protein obtained with the respective enzyme. However, TrPepz was more efficient than trypsin at hydrolyzing the C bonds of two or more consecutive lysine and/or arginine residues. Like its esterase activity, the proteolytic activity of ChPepz was inhibited by either DIFP or TPCK whereas that of TrPepz was inhibited by either DIFP or phenylmethylsulfonyl fluoride but not by TPCK. Finally, ChPepz and TrPepz were each more active at low temperature than the respective enzyme. This ability to construct fully functional peptide enzymes (pepzymes) of chosen specificities should find many practical applications.

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