Solid-State Nuclear Magnetic Resonance Studies Delineate the Role of the Protein in Activation of Both Aromatic Rings of Thiamin

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• 作者：Anand Balakrishnan ; Sivakumar Paramasivam ; Sumit Chakraborty ; Tatyana Polenova ; Frank Jordan
• 刊名：The Journal of the American Chemical Society
• 出版年：2012
• 出版时间：January 11, 2012
• 年：2012
• 卷：134
• 期：1
• 页码：665-672
• 全文大小：961K
• 年卷期：v.134,no.1(January 11, 2012)
• ISSN：1520-5126

文摘

Knowledge of the state of ionization and tautomerization of heteroaromatic cofactors when enzyme-bound is essential for formulating a detailed stepwise mechanism via proton transfers, the most commonly observed contribution to enzyme catalysis. In the bifunctional coenzyme, thiamin diphosphate (ThDP), both aromatic rings participate in catalysis, the thiazolium ring as an electrophilic covalent catalyst and the 4鈥?aminopyrimidine as acid鈥揵ase catalyst involving its 1鈥?4鈥?iminopyrimidine tautomeric form. Two of four ionization and tautomeric states of ThDP are well characterized via circular dichroism spectral signatures on several ThDP superfamily members. Yet, the method is incapable of providing information about specific proton locations, which in principle may be accessible via NMR studies. To determine the precise ionization/tautomerization states of ThDP during various stages of the catalytic cycle, we report the first application of solid-state NMR spectroscopy to ThDP enzymes, whose large mass (160,000鈥?50,000 Da) precludes solution NMR approaches. Three de novo synthesized analogues, [C2,C6鈥?¹³C₂]ThDP, [C2-¹³C]ThDP, and [N4鈥?¹⁵N]ThDP used with three enzymes revealed that (a) binding to the enzymes activates both the 4鈥?aminopyrimidine (via pK_a elevation) and the thiazolium rings (pK_a suppression); (b) detection of a pre-decarboxylation intermediate analogue using [C2,C6鈥?¹³C₂]ThDP, enables both confirmation of covalent bond formation and response in 4鈥?aminopyrimidine ring鈥檚 tautomeric state to intermediate formation, supporting the mechanism we postulate; and (c) the chemical shift of bound [N4鈥?¹⁵N]ThDP provides plausible models for the participation of the 1鈥?4鈥?iminopyrimidine tautomer in the mechanism. Unprecedented detail is achieved about proton positions on this bifunctional coenzyme on large enzymes in their active states.