Pecoraro, V.L.

Angew. Chem. Int. Ed. 2014, 53, 7900-7903, 10.1002/anie.201404925

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 ZnII metalloenzyme was prepared using de novo design. α3DH3 folds into a stable single‐stranded three‐helix bundle and binds ZnII with high affinity using His3O coordination. The resulting metalloenzyme catalyzes the hydration of CO2 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.

Roelfes, G.

Chem. Sci. 2013, 4, 3578, 10.1039/c3sc51449h

Direct addition of water to alkenes to generate important chiral alcohols as key motif in a variety of natural products still remains a challenge in organic chemistry. Here, we report the first enantioselective artificial metallo-hydratase, based on the transcription factor LmrR, which catalyses the conjugate addition of water to generate chiral β-hydroxy ketones with enantioselectivities up to 84% ee. A mutagenesis study revealed that an aspartic acid and a phenylalanine located in the active site play a key role in achieving efficient catalysis and high enantioselectivities.

Pecoraro, V.L.

Nat. Chem. 2012, 4, 118-123, 10.1038/NCHEM.1201

Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions—a Zn(II) ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency only ~100-fold less than that of human carbonic anhydrase (CA)II and at least 550-fold better than comparable synthetic complexes. Similarly, CO2 hydration occurs with an efficiency within ~500-fold of CAII. Although histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO2 hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals.

Coleman, J.E.

Nature 1967, 214, 193-194, 10.1038/214193a0

ACETAZOLAMIDE (2-acetylamino-1,3,4-thiadiazole-5-sulphonamide, ‘Diamox’) is the most potent known inhibitor of the zinc enzyme carbonic anhydrase. This communication reports the direct demonstration that binding of acetazolamide to human carbonic anhydrase requires the presence of a metal ion at the active site and that binding depends on the species of divalent metal ion present. Zinc (II) and cobalt (II) ions are the only ions which induce the formation of very stable acetazolamide carbonic anhydrase complexes and are also the ions which most effectively catalyse the hydration of carbon dioxide and the hydrolysis of p-nitrophenyl acetate. Metal-binding monodentate ions, CN−, HS−, OCN−, and N3−, known as effective carbonic anhydrase inhibitors, compete for the acetazolamide binding site of the zinc enzyme.