A De Novo Designed Metalloenzyme for the Hydration of CO 2

Protein design will ultimately allow for the creation of artificial enzymes with novel functions and unprecedented stability. To test our current mastery of nature’s approach to catalysis, a Zn II metalloenzyme was prepared using de novo design. α 3 DH 3 folds into a stable single‐stranded three‐helix bundle and binds Zn II with high affinity using His 3 O coordination. The resulting metalloenzyme catalyzes the hydration of CO 2 better than any small molecule model of carbonic anhydrase and with an efficiency within 1400‐fold of the fastest carbonic anhydrase isoform, CAII, and 11‐fold of CAIII. Chasing down the cheetah : A synthetic metalloenzyme was created that is capable of catalyzing the hydration of carbon dioxide with an efficiency within 1400‐fold of carbonic anhydrase II, one of the most efficient enzymes known. This designed zinc enzyme performs better than small‐molecule models of carbonic anhydrase. Picture: Zn purple, N dark blue, O red, C cyan.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108029/1/anie_201404925_sm_miscellaneous_information.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/108029/2/7900_ftp.pd

Mechanically activated rupture of single covalent bonds: evidence of force induced bond hydrolysis.

We have used temperature-dependent single molecule force spectroscopy to stretch covalently anchored carboxymethylated amylose (CMA) polymers attached to an amino-functionalized AFM cantilever. Using an Arrhenius kinetics model based on a Morse potential as a one-dimensional representation of covalent bonds, we have extracted kinetic and structural parameters of the bond rupture process. With 35.5 kJ mol−1, we found a significantly smaller dissociation energy and with 9.0 × 102 s−1 to 3.6 × 103 s−1 also smaller Arrhenius pre-factors than expected for homolytic bond scission. One possible explanation for the severely reduced dissociation energy and Arrhenius pre-factors is the mechanically activated hydrolysis of covalent bonds. Both the carboxylic acid amide and the siloxane bond in the amino-silane surface linker are in principle prone to bond hydrolysis. Scattering, slope and curvature of the scattered data plots indicate that in fact two competing rupture mechanisms are observed

Sculpting Metal‐binding Environments in De Novo Designed Three‐helix Bundles

De novo protein design is a biologically relevant approach used to study the active centers of native metalloproteins. In this review, we will first discuss the design process in achieving α3D, a de novo designed three‐helix bundle peptide with a well‐defined fold. We will then cover our recent work in functionalizing the α3D framework by incorporating a tris(cysteine) and tris(histidine) motif. Our first design contains the thiol‐rich sites found in metalloregulatory proteins that control the levels of toxic metal ions (Hg, Cd, and Pb). The latter design recapitulates the catalytic site and activity of a natural metalloenzyme carbonic anhydrase. The review will conclude with future design goals aimed at introducing an asymmetric metal‐binding site in the α3D framework.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110624/1/85_ftp.pd