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Cloning, expression and some properties of -carbonic anhydrase from Helicobacter pylori
Umeå University, Faculty of Science and Technology, Chemistry.
Umeå University, Faculty of Science and Technology, Chemistry.
Umeå University, Faculty of Science and Technology, Chemistry.
2001 (English)In: Biochimica et Biophysica Acta (BBA)/Protein Structure and Molecular Enzymology, Vol. 1544, no 1, 55-63 p.Article in journal (Refereed) Published
Abstract [en]

The -carbonic anhydrase gene from Helicobacter pylori strain 26695 has been cloned and sequenced. The full-length protein appears to be toxic to Escherichia coli, so we prepared a modified form of the gene lacking a part that presumably encodes a cleavable signal peptide. This truncated gene could be expressed in E. coli yielding an active enzyme comprising 229 amino acid residues. The amino acid sequence shows 36% identity with that of the enzyme from Neisseria gonorrhoeae and 28% with that of human carbonic anhydrase II. The H. pylori enzyme was purified by sulfonamide affinity chromatography and its circular dichroism spectrum and denaturation profile in guanidine hydrochloride have been measured. Kinetic parameters for CO2 hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25oC are kcat=2.4x105 s-1, KM=17 mM and kcat/KM=1.4x107 M-1 s-1. The pH dependence of kcat/KM fits with a simple titration curve with pKa=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO2 hydration is limited by proton transfer between a zinc-bound water molecule and the reaction medium in analogy to other forms of the enzyme. The 4-nitrophenyl acetate hydrolase activity of the H. pylori enzyme is quite low with an apparent catalytic second-order rate constant, kenz, of 24 M-1 s-1 at pH 8.8 and 25oC. However, with 2-nitrophenyl acetate as substrate a kenz value of 665 M-1 s-1 was obtained under similar conditions.

Place, publisher, year, edition, pages
2001. Vol. 1544, no 1, 55-63 p.
Keyword [en]
Carbonic anhydrase, Circular dichroism, Enzyme kinetics, Denaturation profile, Helicobacter pylori
URN: urn:nbn:se:umu:diva-8770DOI: doi:10.1016/S0167-4838(00)00204-1OAI: diva2:148441
Available from: 2008-02-11 Created: 2008-02-11 Last updated: 2011-01-13Bibliographically approved

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