Allosteric site variants of Haemophilus influenzae beta-carbonic anhydrase.
Haemophilus influenzae beta-carbonic anhydrase (HICA) is hypothesized to be an allosteric protein that is regulated by the binding of bicarbonate ion to a non-catalytic (inhibitory) site that controls the ligation of Asp44 to the catalytically essential zinc ion. We report here the X-ray crystallographic structures of two variants (W39F and Y181F) involved in the binding of bicarbonate ion in the non-catalytic site and an active-site variant (D44N) that is incapable of forming a strong zinc ligand. The alteration of Trp39 to Phe increases the apparent K(i) for bicarbonate inhibition by 4.8-fold. While the structures of W39F and Y181F are very similar to the wild-type enzyme, the X-ray crystal structure of the D44N variant reveals that it has adopted an active-site conformation nearly identical to that of non-allosteric beta-carbonic anhydrases. We propose that the structure of the D44N variant is likely to be representative of the active conformation of the enzyme. These results lend additional support to the hypothesis that HICA is an allosteric enzyme that can adopt active and inactive conformations, the latter of which is stabilized by bicarbonate ion binding to a non-catalytic site.
Rowlett
Roger S
RS
Department of Chemistry, Colgate University, 13 Oak Drive, Hamilton, New York 13346, USA. rrowlett@mail.colgate.edu
Tu
Chingkuang
C
Lee
Joseph
J
Herman
Ariel G
AG
Chapnick
Douglas A
DA
Shah
Shalini H
SH
Gareiss
Peter C
PC
D000495:Allosteric Site / Q000235:genetics*; D019943:Amino Acid Substitution; D001639:Bicarbonates / Q000737:chemistry; D055162:Biocatalysis; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010103:Oxygen Isotopes / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D001639:Bicarbonates; D010103:Oxygen Isotopes; D011994:Recombinant Proteins; D002256:Carbonic Anhydrases; D015032:Zinc