Cloning, expression and some properties of α-carbonic anhydrase from Helicobacter pylori

Abstract

The α-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 CO 2 hydration catalyzed by the H. pylori enzyme at pH 8.9 and 25°C are k cat=2.4×10 5 s −1, K M=17 mM and k cat/ K M=1.4×10 7 M −1 s −1. The pH dependence of k cat/ K M fits with a simple titration curve with p K a=7.5. Thiocyanate yields an uncompetitive inhibition pattern at pH 9 indicating that the maximal rate of CO 2 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, k enz, of 24 M −1 s −1 at pH 8.8 and 25°C. However, with 2-nitrophenyl acetate as substrate a k enz value of 665 M −1 s −1 was obtained under similar conditions.