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 N eisseria gonorrhoeae has been determined. The gene encodes a 252-resid ue polypeptide with a molecular mass of 28 085 Da. The gene has been c loned and overexpressed in Escherichia coli, and the enzyme has been p urified. A 26-residue signal peptide is cleaved off by the E. coli pro cessing machinery. Thus, the isolated enzyme contains 226 amino acid r esidues with a molecular mass of 25 314 Da. Most of the enzyme seems t o be produced as a soluble protein located in the periplasm of E. coli . The enzyme is homologous to carbonic anhydrases from the animal king dom; it is an alpha-carbonic anhydrase. A comparison with the amino ac id sequences of human carbonic anhydrases I and II suggests that the s econdary 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-acti vity human isozyme II. The bacterial enzyme has a high CO2 hydration a ctivity with a k(cat) of 1.1 . 10(6) s(-1) and K-m of 20 mM at pH 9 an d 25 degrees C. The enzyme also catalyzes the hydrolysis of 4-nitrophe nyl acetate. The pH/rate profile can be described as a titration curve with pK(a) of 6.7 and a maximal value of the catalytic second-order r ate constant, k(enz), of 130 M(-1). s(-1).