Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the γ class of carbonic anhydrases

Sabrina A. Zimmerman, Jean Francois Tomb, James G. Ferry

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

The homotrimeric enzyme Mt-Cam from Methanosarcina thermophila is the archetype of the γ class of carbonic anhydrases. A search of databases queried with Mt-Cam revealed that a majority of the homologs comprise a putative subclass (CamH) in which there is major conservation of all of the residues essential for the archetype Mt-Cam except Glu62 and an acidic loop containing the essential proton shuttle residue Glu84. The CamH homolog from M. thermophila (Mt-CamH) was overproduced in Escherichia coli and characterized to validate its activity and initiate an investigation of the CamH subclass. The Mt-CamH homotrimer purified from E. coli cultured with supplemental zinc (Zn-Mt-CamH) contained 0.71 zinc and 0.15 iron per monomer and had kcat and k cat/Km values that were substantially lower than those for the zinc form of Mt-Cam (Zn-Mt-Cam). Mt-CamH purified from E. coli cultured with supplemental iron (Fe-Mt-CamH) was also a trimer containing 0.15 iron per monomer and only a trace amount of zinc and had an effective kcat (kcateff) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the kcat/K meff was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the kcateff of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H2O2, which supports the hypothesis that Fe 2+ has a role in the active site. The kcat for Fe-Mt-CamH was dependent on the concentration of buffer in a way that indicates that it acts as a second substrate in a "ping-pong" mechanism accepting a proton. The kcat/Km was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO2 and HCO3- is separate from intermolecular proton transfer.

Original languageEnglish (US)
Pages (from-to)1353-1360
Number of pages8
JournalJournal of bacteriology
Volume192
Issue number5
DOIs
StatePublished - Mar 2010

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

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