Kinetic and Structural Characterization of Thermostabilized Mutants of Human Carbonic Anhydrase II

Overview

Journal Protein Eng Des Sel

Publisher Oxford University Press

Specialties Biochemistry
Biotechnology

Date 2012 Jun 14

PMID 22691706

Citations 13

Authors

Zoe Fisher

Christopher D Boone

Shya Masri Biswas

Balasubramanian Venkatakrishnan

Mayank Aggarwal

Chingkuang Tu

Mavis Agbandje-Mckenna

David Silverman

Robert McKenna

Affiliations

Soon will be listed here.

Abstract

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. As such, there is enormous industrial interest in using CA as a bio-catalyst for carbon sequestration and biofuel production. However, to ensure cost-effective use of the enzyme under harsh industrial conditions, studies were initiated to produce variants with enhanced thermostability while retaining high solubility and catalytic activity. Kinetic and structural studies were conducted to determine the structural and functional effects of these mutations. X-ray crystallography revealed that a gain in surface hydrogen bonding contributes to stability while retaining proper active site geometry and electrostatics to sustain catalytic efficiency. The kinetic profiles determined under a variety of conditions show that the surface mutations did not negatively impact the carbon dioxide hydration or proton transfer activity of the enzyme. Together these results show that it is possible to enhance the thermal stability of human carbonic anhydrase II by specific replacements of surface hydrophobic residues of the enzyme. In addition, combining these stabilizing mutations with strategic active site changes have resulted in thermostable mutants with desirable kinetic properties.

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