Structural Basis for the Catalytic Mechanism of Mammalian 25-kDa Thiamine TriphosphataseS
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 t...
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| Published in | The Journal of biological chemistry Vol. 283; no. 16; pp. 10939 - 10948 |
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| Main Authors | , , , |
| Format | Journal Article |
| Language | English |
| Published |
American Society for Biochemistry and Molecular Biology
18.04.2008
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| Subjects | |
| Online Access | Get full text |
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| Summary: | 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 Mg
2+
and ThTP
were determined from NMR titration experiments. The binding of Mg
2+
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·Mg
2+
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 Mg
2+
in catalysis. We propose a mechanism
underlying the high substrate specificity of mThTPase and discuss the possible
role of water molecules in enzymatic catalysis. |
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| Bibliography: | The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S2. 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. 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@nmrfam.wisc.edu. The Research Director of the F.R.S-FNRS. |
| ISSN: | 0021-9258 1083-351X |
| DOI: | 10.1074/jbc.M709675200 |