title: A Chemically Programmed Proximal Ligand Enhances the Catalytic Properties of a Heme Enzyme.
abstract: Enzymes rely on complex interactions between precisely positioned active site residues as a mechanism to compensate for the limited functionality contained within the genetic code. Heme enzymes provide a striking example of this complexity, whereby the electronic properties of reactive ferryl intermediates are finely tuned through hydrogen bonding interactions between proximal ligands and neighboring amino acids. Here, we show that introduction of a chemically programmed proximal Nδ-methyl histidine (NMH) ligand into an engineered ascorbate peroxidase (APX2) overcomes the reliance on the conserved Asp-His hydrogen bonding interaction, leading to a catalytically modified enzyme (APX2 NMH), which is able to achieve a significantly higher number of turnovers compared with APX2 without compromising catalytic efficiency. Structural, spectroscopic and kinetic characterization of APX2 NMH and several active site variants provides valuable insights into the role of the Asp-His-Fe triad of heme peroxidases. More significantly, simplification of catalytic mechanisms through the incorporation of chemically optimized ligands may facilitate efforts to create and evolve new active site heme environments within proteins.
authors: School of Chemistry & Manchester Institute of Biotechnology, The University of Manchester , 131 Princess Street, Manchester M1 7DN, U.K.
authors: Anthony P
authors: AP
authors: Green
authors: Laboratory of Organic Chemistry, ETH Zurich , 8093 Zürich, Switzerland.
authors: Takahiro
authors: T
authors: Hayashi
authors: Department of Biochemistry, University of Zürich , 8057 Zürich, Switzerland.
authors: Peer R E
authors: PR
authors: Mittl
authors: Laboratory of Organic Chemistry, ETH Zurich , 8093 Zürich, Switzerland.
authors: Donald
authors: D
authors: Hilvert
mesh_terms: D060387:Ascorbate Peroxidases / Q000737:chemistry* / Q000378:metabolism*; D055162:Biocatalysis; D017124:Conserved Sequence; D006418:Heme / Q000378:metabolism*; D006860:Hydrogen Bonding; D008024:Ligands; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation
chemical_list: D008024:Ligands; D006418:Heme; D060387:Ascorbate Peroxidases