Cyclic peptide formation catalyzed by an antibody ligase

David B. Smithrud, Patricia A. Benkovic, Stephen J. Benkovic, Victoria Roberts, Josephine Liu, Irina Neagu, Seiji Iwama, Barton W. Phillips, Amos B. Smith, Ralph Hirschmann

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15 Scopus citations

Abstract

Cyclic hexapeptides represent a class of compounds with important, diverse biological activities. We report herein that the antibody 16G3 catalyzes the cyclization of D-Trp-Gly-Pal-Pro-Gly-Phe·p-nitrophenyl ester (8a) to give C-(D-Trp-Gly-Pal-Pro-Gly-L-Phe) (11a). The antibody does not, however, catalyze either epimerization or hydrolysis. The resulting rate enhancement of the cyclization by 16G3 (22-fold) was sufficient to form the desired product in greater than 90% yield. In absolute rate terms, the turnover of 16G3 is estimated to be 2 min-1. The background rate of epimerization of 8a was reduced from 10 to 1% and hydrolysis from 50 to 4% in the presence of 16G3. As expected, the catalytic effects of 16G3 were blocked by the addition of an amount of the hapten equal to twice the antibody concentration. We also synthesized three diastereomers of 8a: the D-Trp1-D- Phe6 (8b), L-Trp1-L-Phe6 (8C), and L-Trp1-D-Phe6 (8d) hexapeptides as well as D-Trp'-L-Trp6 (12) and D-Phe'-L-Phe6 (13). As expected, the rate enhancement by 16G3 was greatest for 8a, because the stereochemistry of Trp1 and Phe6 matches that of the corresponding residues on the hapten used to induce the biosynthesis of 16G3. A model of the variable domain of 16G3 was generated from the primary sequence using the antibody structural database to guide the model construction. The resulting model provided support for some previously proposed interpretations of the kinetic data, while providing valuable new insights for others.

Original languageEnglish (US)
Pages (from-to)1953-1958
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume97
Issue number5
DOIs
StatePublished - Feb 29 2000

All Science Journal Classification (ASJC) codes

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    Smithrud, D. B., Benkovic, P. A., Benkovic, S. J., Roberts, V., Liu, J., Neagu, I., Iwama, S., Phillips, B. W., Smith, A. B., & Hirschmann, R. (2000). Cyclic peptide formation catalyzed by an antibody ligase. Proceedings of the National Academy of Sciences of the United States of America, 97(5), 1953-1958. https://doi.org/10.1073/pnas.040534397