Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the {gamma} class of carbonic anhydrases.
The homotrimeric enzyme Mt-Cam from Methanosarcina thermophila is the archetype of the gamma 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 k(cat) and k(cat)/K(m) 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 k(cat) (k(cat)(eff)) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the k(cat)/K(m)(eff) was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the k(cat)(eff) of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H(2)O(2), which supports the hypothesis that Fe(2+) has a role in the active site. The k(cat) 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 k(cat)/K(m) was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO(2) and HCO(3)(-) is separate from intermolecular proton transfer.
Zimmerman
Sabrina A
SA
Department of Biochemistry and Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, PA 16802-4500, USA.
Tomb
Jean-Francois
JF
Ferry
James G
JG
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003067:Coenzymes / Q000032:analysis; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007501:Iron / Q000032:analysis; D007700:Kinetics; D017020:Methanosarcina / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D015032:Zinc / Q000032:analysis
D019843:Archaeal Proteins; D003067:Coenzymes; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D007501:Iron; D002256:Carbonic Anhydrases; D015032:Zinc