HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 283, Issue 16, 10939-10948, April 18, 2008

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 283/16/10939    most recent M709675200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Song, J. Articles by Markley, J. L. PubMed PubMed Citation Articles by Song, J. Articles by Markley, J. L. Social Bookmarking                      What's this?

Structural Basis for the Catalytic Mechanism of Mammalian 25-kDa Thiamine Triphosphatase^*

Jikui Song, Lucien Bettendorff¹, Marco Tonelli^¶, and John L. Markley^¶2

From the Center for Eukaryotic Structural Genomics and ^¶National Magnetic Resonance Facility at Madison, Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706-1544 and the Center for Cellular and Molecular Neurobiology, University of Liège, Avenue de l'Hôpital 1, B-4000 Liège, Belgium

Mammalian soluble thiamine triphosphatase (ThTPase) is a 25-kDa cytosolic enzyme that specifically catalyzes the conversion of thiamine triphosphate (ThTP) to thiamine diphosphate and has an absolute requirement for divalent cations. We have investigated the kinetic properties of recombinant mouse thiamine triphosphatase (mThTPase) and determined its solution structure by NMR spectroscopy. Residues responsible for binding Mg²⁺ and ThTP were determined from NMR titration experiments. The binding of Mg²⁺ induced only a minor local conformational change, whereas ThTP binding was found to cause a more global conformational change. We derived a structural model for the mThTPase·ThTP·Mg²⁺ ternary complex and concluded from this that whereas free mThTPase has an open cleft fold, the enzyme in the ternary complex adopts a tunnel fold. Our results provide a functional rationale for a number of conserved residues and suggest an essential role for Mg²⁺ in catalysis. We propose a mechanism underlying the high substrate specificity of mThTPase and discuss the possible role of water molecules in enzymatic catalysis.

Received for publication, November 27, 2007 , and in revised form, February 6, 2008.

The atomic coordinates and structure factors (code 2JMU) 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 grants from the National Institutes of Health, Protein Structure Initiative through Grants P50 GM64598 and U54 GM074901 (to J. L. M.) and the Fonds de la Recherche Scientifique FNRS (F.R.S.-FNRS) through Grant 2.4.558.04.F (to L. B.). NMR data were collected at the National Magnetic Resonance Facility at Madison, which is supported in part by National Institutes of Health Grants P41 RR02301 and P41 GM66326 (to J. L. M.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S2.

¹ The Research Director of the F.R.S-FNRS.

² To whom correspondence may be addressed, Biochemistry Dept., University of Wisconsin-Madison, 433 Babcock Dr., Madison WI 53706. Tel.: 608-263-9349; Fax: 608-262-3759; E-mail: markley{at}nmrfam.wisc.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?