A closer look at the active site of gamma-class carbonic anhydrases: High-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila
Tm. Iverson et al., A closer look at the active site of gamma-class carbonic anhydrases: High-resolution crystallographic studies of the carbonic anhydrase from Methanosarcina thermophila, BIOCHEM, 39(31), 2000, pp. 9222-9231
The prototype of the gamma-class of carbonic anhydrase has been characteriz
ed from the methanogenic archaeon Methanosarcina thermophila. Previously re
ported kinetic studies of the gamma-class carbonic anhydrase are consistent
with this enzyme having a reaction mechanism similar to that of the mammal
ian alpha-class carbonic anhydrase. However, the overall folds of these two
enzymes are dissimilar, and apart from the zinc-coordinating histidines, t
he active site residues bear little resemblance to one another. The crystal
structures of zinc-containing and cobalt-substituted gamma-class carbonic
anhydrases from M. thermophila are reported here between 1.46 and 1.95 Angs
trom resolution in the unbound form and cocrystallized with either SO42- Or
HCO3-. Relative to the tetrahedral coordination geometry seen at the activ
e site in the alpha-class of carbonic anhydrases, the active site of the ga
mma-class enzyme contains additional metal-bound water ligands, so the over
all coordination geometry is trigonal bipyramidal for the zinc-containing e
nzyme and octahedral for the cobalt-substituted enzyme. Ligands bound to th
e active site all make contacts with the side chain of Glu 62 in manners th
at suggest the side chain is likely protonated. In the uncomplexed zinc-con
taining enzyme, the side chains of Glu 62 and Glu 84 appear to share a prot
on; additionally, Glu 84 exhibits multiple conformations. This suggests tha
t Glu 84 may act as a proton shuttle, which is an important aspect of the r
eaction mechanism of alpha-class carbonic anhydrases. A hydrophobic pocket
on the surface of the enzyme may participate in the trapping of CO2 at the
active site. On the basis of the coordination geometry at the active site,
ligand binding modes, the behavior of the side chains of Glu 62 and Glu 84,
and analogies to the well-characterized alpha-class of carbonic anhydrases
, a more-defined reaction mechanism is proposed for the gamma-class of carb
onic anhydrases.