De novo design of catalytic proteins

doi:10.1073/pnas.0404387101

De novo design of catalytic proteins

J. Kaplan* and
W. F. DeGrado*,†,‡

^*Department of Biochemistry and Biophysics, School of Medicine, and ^†Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6059

Contributed by W. F. DeGrado, June 18, 2004

Abstract

The de novo design of catalytic proteins provides a stringent test of our understanding of enzyme function, while simultaneously laying the groundwork for the design of novel catalysts. Here we describe the design of an O₂-dependent phenol oxidase whose structure, sequence, and activity are designed from first principles. The protein catalyzes the two-electron oxidation of 4-aminophenol (k _cat/K _M = 1,500 M^·1·min^·1) to the corresponding quinone monoimine by using a diiron cofactor. The catalytic efficiency is sensitive to changes of the size of a methyl group in the protein, illustrating the specificity of the design.

Footnotes

↵ ‡ To whom correspondence should be addressed. E-mail: wdegrado{at}mail.med.upenn.edu.
Abbreviations: DF, dueferri; 4AP, 4-aminophenol; 4CP, 4-cyanophenol; MPD, 3-phenylenediamine.
Note Added in Proof. While this paper was in review, Hellinga and coworkers (25) described and used computational methods to predict mutations that introduce triose phosphate isomerase activity into ribose-binding protein, a receptor that normally lacks enzyme activity.