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Structural Characterization of the Enzyme−Substrate, Enzyme−Intermediate, and Enzyme−Product Complexes of Thiamin Phosphate Synthase

Peapus, Diane H. and Chiu, Hsiu-Ju and Campobasso, Nino and Reddick, Jason J. and Begley, Tadhg P. and Ealick, Steven E. (2001) Structural Characterization of the Enzyme−Substrate, Enzyme−Intermediate, and Enzyme−Product Complexes of Thiamin Phosphate Synthase. Biochemistry, 40 (34). pp. 10103-10114. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20170131-102251279

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Abstract

Thiamin phosphate synthase catalyzes the formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxyethyl)-4-methylthiazole phosphate. Several lines of evidence suggest that the reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We report here seven new crystal structures primarily involving crystals of S130A thiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130A thiamin phosphate synthase with the intent of characterizing the enzyme−substrate complex. Surprisingly, in three thiamin phosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the electron density is provided by a model that consists of independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbenium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe is the species that is observed in the crystal structures.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/bi0104726DOIArticle
http://pubs.acs.org/doi/full/10.1021/bi0104726PublisherArticle
Additional Information:© 2001 American Chemical Society. Received 8 March 2001 .Published online 21 July 2001. Published in print 1 August 2001. This work was supported by National Institutes of Health grants to T.P.B. (DK44083) and by Hoffmann-La Roche (T.P.B.). S.E.E. is indebted to the W. M. Keck Foundation and the Lucille P. Markey Charitable Trust. We thank Ms. Leslie Kinsland for assistance in the preparation of the manuscript. We thank CHESS and the Structural Biology Center at the APS for providing beam time.
Funders:
Funding AgencyGrant Number
NIHDK44083
Hoffmann-La RocheUNSPECIFIED
W. M. Keck FoundationUNSPECIFIED
Lucille P. Markey Charitable TrustUNSPECIFIED
Issue or Number:34
Record Number:CaltechAUTHORS:20170131-102251279
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20170131-102251279
Official Citation:Structural Characterization of the Enzyme−Substrate, Enzyme−Intermediate, and Enzyme−Product Complexes of Thiamin Phosphate Synthase Diane H. Peapus, Hsiu-Ju Chiu, Nino Campobasso, Jason J. Reddick, Tadhg P. Begley, and Steven E. Ealick Biochemistry 2001 40 (34), 10103-10114 DOI: 10.1021/bi0104726
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:73875
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:31 Jan 2017 18:38
Last Modified:03 Oct 2019 16:32

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