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Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the {gamma} class of carbonic anhydrases.

Abstract：

: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.

journal_name

J Bacteriol

journal_title

Journal of bacteriology

authors

Zimmerman SA,Tomb JF,Ferry JG

doi

10.1128/JB.01164-09

subject

Has Abstract

pub_date

2010-03-01 00:00:00

pages

1353-60

issue

eissn

0021-9193

issn

1098-5530

pii

JB.01164-09

journal_volume

192

pub_type

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Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the {gamma} class of carbonic anhydrases.