Proposal for a hydrogen bond network in the active site of the prototypic gamma-class carbonic anhydrase

Biochemistry
Sabrina Zimmerman, J G Ferry

Abstract

The crystal structure of Cam, the prototypic gamma-class carbonic anhydrase, reveals active site residues Gln75, Asn73, and Asn 202 previously hypothesized to participate in catalysis. These potential roles were investigated for the first time by kinetic analyses of site-specific replacement variants of the zinc and cobalt forms of Cam. Gln75 replacement variants showed large decreases in k(cat)/K(m) relative to wild-type. Further, the Gln75 variants showed a loss of the pK(a) in pH versus k(cat)/K(m) profiles previously attributed to ionization of the metal-bound water yielding the hydroxyl group attacking CO(2). These results support the previously proposed role for Gln75 in hydrogen bonding with the catalytic hydroxyl orienting it for attack on CO(2). Kinetic analyses of Asn73 variants were consistent with a role in hydrogen bonding with Gln75 to position it for optimal interaction with the catalytic hydroxyl. Kinetic analyses of Asn202 variants showed substantial decreases in k(cat)/K(m) relative to the wild-type enzyme supporting the previously hypothesized role in polarizing CO(2) and facilitating attack from the metal-bound hydroxyl. On the basis of results presented here, and previously reported structural analyses, we ...Continue Reading

References

Jan 1, 1997·Pharmacology & Therapeutics·S Lindskog
Nov 14, 2000·Journal of Bacteriology·K S SmithJ G Ferry
Jun 19, 2002·Journal of Theoretical Biology·Silke Thoms

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Citations

May 7, 2008·The Protein Journal·Saule NaureckienePranab K Chanda
Feb 6, 2010·Proceedings of the National Academy of Sciences of the United States of America·Kerry L PeñaMatthew S Kimber
Jun 10, 2010·Annual Review of Microbiology·James G Ferry
Jan 24, 2013·Dalton Transactions : an International Journal of Inorganic Chemistry·Li ZhangDian-Zeng Jia
Jul 18, 2015·International Journal of Molecular Sciences·Anna Di FioreKatia D'Ambrosio
Nov 1, 2012·Archives of Biochemistry and Biophysics·Sabrina ZimmermanJames G Ferry
Sep 15, 2009·Biochimica Et Biophysica Acta·James G Ferry
Apr 2, 2008·Annals of the New York Academy of Sciences·James G Ferry, Daniel J Lessner

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