title: Role of an evolutionarily invariant serine for the stability of human carbonic anhydrase II.
abstract: There are several evolutionarily invariant amino acids in the primary structures of all known isoenzymes of carbonic anhydrase. One of these is Ser-29 which is situated in the peripheral part of the active site interacting by hydrogen bonds with amino acids located nearby in the tertiary structure. Furthermore, the neighbourhood of Ser-29, composed of Gln-28, Pro-30, Tyr-194, Ser-197 and Trp-209, has a totally invariant structure. The structural role of Ser-29 was investigated by site-directed mutagenesis. The stability of two enzyme mutants, where Ser-29 was replaced by alanine and cysteine, towards denaturation by guanidine-HCl was studied. Changing Ser-29 to Ala resulted in a destabilization by 2.6 kcal/mol, corresponding to the loss of 2-3 hydrogen bonds. Interestingly, Ser-29 is within hydrogen bond distance to Tyr-194, Ser-197 and Trp-209 in the tertiary structure. Therefore, rupture of these interactions caused by the Ser-29----Ala substitution could explain the observed destabilization of this enzyme variant. Substituting cysteine for Ser-29 gives rise to a drastic decrease in the stability of the protein (change in midpoint concentration of denaturation from 0.96 M to less than 0.1 M guanidine-HCl) despite the minor structural change (O----S atom). This destabilization corresponds to approx. 7-8 kcal/mol and cannot be explained by changes in hydrogen bond pattern only, but must also include unfavourable conformational changes to avoid van der Waals collisions originating from the somewhat larger thiol group.
authors.affiliation: Department of Biochemistry, Umeå University, Sweden.
authors.forename: L G
authors.initials: LG
authors.lastname: Mårtensson
authors.forename: B H
authors.initials: BH
authors.lastname: Jonsson
authors.forename: M
authors.initials: M
authors.lastname: Andersson
authors.forename: A
authors.initials: A
authors.lastname: Kihlgren
authors.forename: N
authors.initials: N
authors.lastname: Bergenhem
authors.forename: U
authors.initials: U
authors.lastname: Carlsson
mesh_terms: D000409:Alanine / Q000235:genetics; D001665:Binding Sites; D005075:Biological Evolution; D002256:Carbonic Anhydrases / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D009154:Mutation; D011487:Protein Conformation; D012694:Serine / Q000235:genetics* / Q000378:metabolism
chemical_list: D012694:Serine; D002256:Carbonic Anhydrases; D003545:Cysteine; D000409:Alanine