The complete sequence, expression in Escherichia coli, purification and some properties of carbonic anhydrase from Neisseria gonorrhoeae.
The complete nucleotide sequence of the carbonic anhydrase gene from Neisseria gonorrhoeae has been determined. The gene encodes a 252-residue polypeptide with a molecular mass of 28085 Da. The gene has been cloned and overexpressed in Escherichia coli, and the enzyme has been purified. A 26-residue signal peptide is cleaved off by the E. coli processing machinery. Thus, the isolated enzyme contains 226 amino acid residues with a molecular mass of 25314 Da. Most of the enzyme seems to be produced as a soluble protein located in the periplasm of E. coli. The enzyme is homologous to carbonic anhydrases from the animal kingdom; it is an alpha-carbonic anhydrase. A comparison with the amino acid sequences of human carbonic anhydrases I and II suggests that the secondary structures are essentially intact in the bacterial enzyme but that several loops are much shorter than in the human forms. Most of the active-site residues are identical to those found in the high-activity human isozyme II. The bacterial enzyme has a high CO2 hydration activity with a k(cat) of 1.1 x 10(6) s(-1) and Km of 20 mM at pH 9 and 25 degrees C. The enzyme also catalyzes the hydrolysis of 4-nitrophenyl acetate. The pH/rate profile can be described as a titration curve with pKa of 6.7 and a maximal value of the catalytic second-order rate constant, k(enz), of 130 M(-1) x s(-1).
Chirică
L C
LC
Department of Biochemistry, Umeå University, Sweden.
Elleby
B
B
Jonsson
B H
BH
Lindskog
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002256:Carbonic Anhydrases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009344:Neisseria gonorrhoeae / Q000201:enzymology* / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D004269:DNA, Bacterial; D002256:Carbonic Anhydrases