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Published December 4, 2001 | Published
Journal Article Open

Enzyme-like proteins by computational design


We report the development and initial experimental validation of a computational design procedure aimed at generating enzyme-like protein catalysts called "protozymes." Our design approach utilizes a "compute and build" strategy that is based on the physical/chemical principles governing protein stability and catalytic mechanism. By using the catalytically inert 108-residue Escherichia coli thioredoxin as a scaffold, the histidine-mediated nucleophilic hydrolysis of p-nitrophenyl acetate as a model reaction, and the ORBIT protein design software to compute sequences, an active site scan identified two promising catalytic positions and surrounding active-site mutations required for substrate binding. Experimentally, both candidate protozymes demonstrated catalytic activity significantly above background. One of the proteins, PZD2, displayed "burst" phase kinetics at high substrate concentrations, consistent with the formation of a stable enzyme intermediate. The kinetic parameters of PZD2 are comparable to early catalytic Abs. But, unlike catalytic Ab design, our design procedure is independent of fold, suggesting a possible mechanism for examining the relationships between protein fold and the evolvability of protein function.

Additional Information

© 2001 by The National Academy of Sciences From the cover Communicated by Douglas C. Rees, California Institute of Technology, Pasadena, CA, October 17, 2001 (received for review August 6, 2001) We acknowledge F. S. Lee and D. Bökenkamp for their important early efforts and P. Strop and R. A. Olofson for helpful discussions. We thank G. Hathaway and the Protein/Peptide Micro Analytical Laboratory for mass spectra. This work was supported by the Howard Hughes Medical Institute and the Ralph M. Parsons Foundation (S.L.M.), the Helen G. and Arthur McCallum Foundation, the Evelyn Sharp Graduate Fellowship, and by a training grant from the National Institutes of Health (D.N.B.). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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