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

被引:49
作者
Chirica, LC [1 ]
Elleby, B [1 ]
Lindskog, S [1 ]
机构
[1] Umea Univ, Dept Biochem, S-90187 Umea, Sweden
来源
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY | 2001年 / 1544卷 / 1-2期
关键词
carbonic anhydrase; circular dichroism; enzyme kinetics; denaturation profile; Helicobacter pylori;
D O I
10.1016/S0167-4838(00)00204-1
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The ct-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 degreesC are k(cat) = 2.4 x 10(5) s(-1), K-M = 17 mM and k(cat)/K-M = 1.4 X 10(7) M-1 s(-1). The pH dependence of k(cat)/K-M 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 s(-1) at pH 8.8 and 25 degreesC, However, with 2-nitrophenyl acetate as substrate a k(enz) value of 665 M (1) s(-1) was obtained under similar conditions. (C) 2001 Elsevier Science B.V. All rights reserved.
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页码:55 / 63
页数:9
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