Cloning, expression and some properties of alpha-carbonic anhydrase from Helicobacter pylori.
The alpha-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 25 degrees C are kcat=2.4x10(5) s(-1), KM=17 mM and kcat/KM=1.4x10(7) M(-1) x s(-1). The pH dependence of kcat/KM fits with a simple titration curve with pK(a)=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, k(enz), of 24 M(-1) x s(-1) at pH 8.8 and 25 degrees C. However, with 2-nitrophenyl acetate as substrate a k(enz) value of 665 M(-1) x s(-1) was obtained under similar conditions.
Chirica
L C
LC
Department of Biochemistry, Umeå University, 90187, Umeå, Sweden.
Elleby
B
B
Lindskog
S
S
D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D016480:Helicobacter pylori / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D002256:Carbonic Anhydrases