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J. Biol. Chem., Vol. 281, Issue 8, 4993-4999, February 24, 2006

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Structural Mechanics of the pH-dependent Activity of -Carbonic Anhydrase from Mycobacterium tuberculosis^*

From the Department of Cell and Molecular Biology, Uppsala University, S-751 24 Uppsala, Sweden

Carbonic anhydrases catalyze the reversible hydration of carbon dioxide to form bicarbonate, a reaction required for many functions, including carbon assimilation and pH homeostasis. Carbonic anhydrases are divided into at least three classes and are believed to share a zinc-hydroxide mechanism for carbon dioxide hydration. -carbonic anhydrases are broadly spread among the domains of life, and existing structures from different organisms show two distinct active site setups, one with three protein coordinations to the zinc (accessible) and the other with four (blocked). The latter is believed to be inconsistent with the zinc-hydroxide mechanism. The Mycobacterium tuberculosis Rv3588c gene, shown to be required for in vivo growth of the pathogen, encodes a -carbonic anhydrase with a steep pH dependence of its activity, being active at pH 8.4 but not at pH 7.5. We have recently solved the structure of this protein, which was a dimeric protein with a blocked active site. Here we present the structure of the thiocyanate complexed protein in a different crystal form. The protein now forms distinct tetramers and shows large structural changes, including a carboxylate shift yielding the accessible active site. This structure demonstrated for the first time that a -carbonic anhydrase can switch between the two states. A pH-dependent dimer to tetramer equilibrium was also demonstrated by dynamic light scattering measurements. The data presented here, therefore, suggest a carboxylate shift on/off switch for the enzyme, which may, in turn, be controlled by a dimer-to-tetramer equilibrium.

Received for publication, October 3, 2005 , and in revised form, November 28, 2005.

The atomic coordinates and structure factors (code 2A5V) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by the Swedish Foundation for Strategic Research, the Swedish Research Council, and the European Commission programs Structural Proteomics in Europe (Grant QLG2-CT-2002-00988) and X-TB (Grant QLRT-2000-02018). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Martin Högbom, Dept. of Cell & Molecular Biology, Uppsala University, Biomedical Center, Box 596, S-751 24 Uppsala, Sweden. Tel.: 46-18-4715062; Fax: 46-18-530396; E-mail: hogbom{at}xray.bmc.uu.se.

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