Open AccessStructural and functional differences between carbonic anhydrase isoenzymes I and II as studied by site‐directed mutagenesis
Author(s) -
BEHRAVAN Gity,
JONASSON Per,
JONSSON BengtHarald,
LINDSKOG Sven
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb16054.x
Subject(s) - enzyme kinetics , isozyme , mutant , esterase , mutagenesis , chemistry , site directed mutagenesis , carbonic anhydrase ii , carbonic anhydrase , biochemistry , enzyme , active site , stereochemistry , amino acid , gene
Site‐specific mutagenesis has been used to replace amino acid residues in the active site of human carbonic anhydrase II with residues characterizing carbonic anhydrases I. Previous studies of [Thr200 → His]isoenzyme II [Behravan, G., Jonsson, B.‐H. & Lindskog, S. (1990) Eur. J. Biochem. 190 , 351–357] showed that His200 is important for the specific catalytic properties of isoenzymes I. In this paper some properties of two single mutants, Asn62 → Val and Asn67 → His, as well as a double mutant, Asn67 → His/Thr200 → His, are described. The results show that neither Val62 nor His67 give rise to isoenzyme‐I‐like properties, while the double mutant behaves like the single mutant with His200. At pH 8.9, the variant with Val62 has a higher value of k cat /K m for CO 2 hydration than unmodified isoenzyme II, whereas the variant with His67 has an enhanced k cat value. The replacement of Asn62 with Val results in a 20% increase of the 4‐nitrophenyl acetate hydrolase activity. For the double mutant, the esterase activity is quite close to that calculated on the assumption that the effects of the two single mutations on the free energy of activation are additive.