Author: ["Melissa L. Zastrow","Anna F. A. Peacock","Jeanne A. Stuckey","Vincent L. Pecoraro"]
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Abstract
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. A designed metalloprotein containing an Hg(II) trithiolate centre that provides structural stability, and a Zn(II) tris histidine centre serving as a catalytic mimic of carbonic anhydrase, is shown to display rates that are comparable to the natural enzyme for ester hydrolysis and CO2 hydration. Cite this article
Zastrow, M., Peacock, A., Stuckey, J. et al. Hydrolytic catalysis and structural stabilization in a designed metalloprotein. Nature Chem 4, 118–123 (2012). https://doi.org/10.1038/nchem.1201 