Open AccessStructural and catalytic characterization of a thermally stable and acid‐stable variant of human carbonic anhydrase II containing an engineered disulfide bond
Author(s) -
Boone Christopher D.,
Habibzadegan Andrew,
Tu Chingkuang,
Silverman David N.,
McKenna Robert
Publication year - 2013
Publication title -
acta crystallographica section d
Language(s) - English
Resource type - Journals
ISSN - 1399-0047
DOI - 10.1107/s0907444913008743
Subject(s) - chemistry , differential scanning calorimetry , catalysis , cysteine , bicarbonate , carbonic anhydrase ii , thermal stability , carbonic anhydrase , kinetics , combinatorial chemistry , enzyme , organic chemistry , physics , thermodynamics , quantum mechanics
The carbonic anhydrases (CAs) are a family of mostly zinc metalloenzymes that catalyze the reversible hydration of CO 2 to bicarbonate and a proton. Recently, there has been industrial interest in utilizing CAs as biocatalysts for carbon sequestration and biofuel production. The conditions used in these processes, however, result in high temperatures and acidic pH. This unfavorable environment results in rapid destabilization and loss of catalytic activity in CAs, ultimately resulting in cost‐inefficient high‐maintenance operation of the system. In order to negate these detrimental industrial conditions, cysteines at residues 23 (Ala23Cys) and 203 (Leu203Cys) were engineered into a wild‐type variant of human CA II (HCAII) containing the mutation Cys206Ser. The X‐ray crystallographic structure of the disulfide‐containing HCAII (dsHCAII) was solved to 1.77 Å resolution and revealed that successful oxidation of the cysteine bond was achieved while also retaining desirable active‐site geometry. Kinetic studies utilizing the measurement of 18 O‐labeled CO 2 by mass spectrometry revealed that dsHCAII retained high catalytic efficiency, and differential scanning calorimetry showed acid stability and thermal stability that was enhanced by up to 14 K compared with native HCAII. Together, these studies have shown that dsHCAII has properties that could be used in an industrial setting to help to lower costs and improve the overall reaction efficiency.