Solid-State NMR and Density Functional Theory Studies of Ionization States of Thiamin

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• 作者：Sivakumar Paramasivam ; Anand Balakrishnan ; Olga Dmitrenko ; Amy Godert ; Tadhg P. Begley ; Frank Jordan ; Tatyana Polenova
• 刊名：Journal of Physical Chemistry B
• 出版年：2011
• 出版时间：February 3, 2011
• 年：2011
• 卷：115
• 期：4
• 页码：730-736
• 全文大小：273K
• 年卷期：v.115,no.4(February 3, 2011)
• ISSN：1520-5207

文摘

Thiamin diphosphate (ThDP) is a key coenzyme in sugar metabolism. The 4鈥?aminopyrimidine ring of ThDP cycles through several ionization and tautomeric states during enzyme catalysis, but it is not fully understood which states are adopted during the individual steps of the catalytic cycle. Thiamin has been synthesized with labels selectively inserted into the C2 and C6鈥?positions, as well as into the amino group, creating [C2, C6鈥?¹³C₂] thiamin and [N4鈥?¹⁵N] thiamin. Magic-angle spinning (MAS) NMR spectroscopy has been employed to record the ¹³C and ¹⁵N chemical shift anisotropy (CSA) tensors for C2, C6鈥? and N4鈥?atoms. Our results indicate that the isotropic chemical shifts as well as the principal components of the ¹³C and ¹⁵N CSA tensors are very sensitive to the protonation states in these compounds and, therefore, permit differentiating between the two ionization states, 4-aminopyrimidine and 4-aminopyrimidinium. Using density functional theory (DFT), we have calculated the magnetic shielding anisotropy tensors of C2, C6鈥? and N4鈥?and found excellent agreement between the computed and the experimental tensors. Our findings indicate that MAS NMR spectroscopy in conjunction with DFT calculations is a sensitive probe of ionization states in the thiamin cofactor. The results of this study will serve as a guide for characterization of ionization and tautomeric states of thiamin in complexes with thiamin-dependent enzymes.