Structural Studies Provide New Insights into the Catalytic Mechanism of Carbonic Anhydrase

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Robert Mckenna - Biochemistry and Molecular Biology
University of Florida

Abstract:
Recent high resolution x-ray crystallography, in combination with catalysis studies, on the zinc metalloenzyme carbonic anhydrase II (CA II) have provided detailed insights into the features of this enzymes catalytic mechanism. High pressure CO₂ and cryo-cooled crystals of CA II have captured the first step of CO₂ hydration, the binding of substrate CO₂, and determined the precise binding interactions of CO₂ for both the holo and apo (without zinc) enzymes. These structures provide insight into the binding of CO₂ in a hydrophobic pocket at the active site, and identify this as a productive binding site by comparison to the structure of complexes of enzyme with competitive inhibitors. These studies also demonstrate that the zinc does not play a critical role in the binding or orientation of CO₂. In addition, structural studies have shown, the prominent proton shuttle residue His64 has specific orientations which appear to enhance proton transfer between the active site and bulk solvent. Moreover, the properties of the imidazole side chain of His64, including its conformations and pK_a, are finely tuned by surrounding residues of the active-site cavity such as Tyr7, Asn62, and Asn67. The structure of a network of ordered solvent molecules located between His64 and the active site are also sensitive to surrounding residues. These findings, when considered together, provide a complete view of the catalytic mechanism of this enzyme and provide insights for proposed future studies that will be presented.