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

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• 作者：Diane H. Peapus ; Hsiu-Ju Chiu ; Nino Campobasso ; Jason J. Reddick ; Tadhg P. Begley ; and Steven E. Ealick
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：August 28, 2001
• 年：2001
• 卷：40
• 期：34
• 页码：10103 - 10114
• 全文大小：666K
• 年卷期：v.40,no.34(August 28, 2001)
• ISSN：1520-4995

文摘

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 previouslydetermined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiaminphosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number ofadditional experiments. We report here seven new crystal structures primarily involving crystals of S130Athiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130Athiamin 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 substratesor products. For these structures, the best fit to the electron density is provided by a model that consistsof independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbeniumion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from thepyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe isthe species that is observed in the crystal structures.