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
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.