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Published August 8, 2000 | public
Journal Article

A Closer Look at the Active Site of γ-Class Carbonic Anhydrases: High-Resolution Crystallographic Studies of the Carbonic Anhydrase from Methanosarcina thermophila

Abstract

The prototype of the γ-class of carbonic anhydrase has been characterized from the methanogenic archaeon Methanosarcina thermophila. Previously reported kinetic studies of the γ-class carbonic anhydrase are consistent with this enzyme having a reaction mechanism similar to that of the mammalian α-class carbonic anhydrase. However, the overall folds of these two enzymes are dissimilar, and apart from the zinc-coordinating histidines, the active site residues bear little resemblance to one another. The crystal structures of zinc-containing and cobalt-substituted γ-class carbonic anhydrases from M. thermophila are reported here between 1.46 and 1.95 Å resolution in the unbound form and cocrystallized with either SO_4^2- or HCO_3^-. Relative to the tetrahedral coordination geometry seen at the active site in the α-class of carbonic anhydrases, the active site of the γ-class enzyme contains additional metal-bound water ligands, so the overall coordination geometry is trigonal bipyramidal for the zinc-containing enzyme and octahedral for the cobalt-substituted enzyme. Ligands bound to the active site all make contacts with the side chain of Glu 62 in manners that suggest the side chain is likely protonated. In the uncomplexed zinc-containing enzyme, the side chains of Glu 62 and Glu 84 appear to share a proton; additionally, Glu 84 exhibits multiple conformations. This suggests that Glu 84 may act as a proton shuttle, which is an important aspect of the reaction mechanism of α-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 α-class of carbonic anhydrases, a more-defined reaction mechanism is proposed for the γ-class of carbonic anhydrases.

Additional Information

© 2000 American Chemical Society. Received February 1, 2000; Revised Manuscript Received May 12, 2000. This work was supported by a grant from the National Institutes of Health to J.G.F. (GM44661). T.M.I. is supported by a NIH training grant (GM07737). We are grateful to C. Fierke, D. Silverman, and R. Cheng for critical reading and helpful discussions, B. Tripp for unpublished results, and H. Schindelin for experimental assistance. This work is based upon research conducted at the Stanford Synchrotron Radiation Laboratory (SSRL), which is funded by the Department of Energy, Office of Basic Energy Sciences.

Additional details

Created:
August 19, 2023
Modified:
October 19, 2023