Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase

Citation
Dh. Peapus et al., Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase, BIOCHEM, 40(34), 2001, pp. 10103-10114
Citations number
23
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
40
Issue
34
Year of publication
2001
Pages
10103 - 10114
Database
ISI
SICI code
0006-2960(20010828)40:34<10103:SCOTEE>2.0.ZU;2-1
Abstract
Thiamin phosphate synthase catalyzes the formation of thiamin phosphate fro m 4-amino-5(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxye thyl)-4-methylthiazole phosphate. Several lines of evidence suggest that th e reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reacti on products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We re port here seven new crystal structures primarily involving crystals of S130 A thiamin phosphate synthase soaked in solutions containing substrates or p roducts. We prepared S130A thiamin phosphate synthase with the intent of ch aracterizing the enzyme-substrate complex. Surprisingly, in three thiamin p hosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the el ectron density is provided by a model that consists of independent pyrimidi ne, pyrophosphate, and thiazole phosphate fragments, consistent with a carb enium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrop hosphate to give the pyrimidine iminemethide, which we believe is the speci es that is observed in the crystal structures.