Deprotonations in the Reaction of Flavin-Dependent Thymidylate Synthase

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• 作者：Frederick W. Stull ; Steffen M. Bernard ; Aparna Sapra ; Janet L. Smith ; Erik R. P. Zuiderweg ; Bruce A. Palfey
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：June 14, 2016
• 年：2016
• 卷：55
• 期：23
• 页码：3261-3269
• 全文大小：491K
• 年卷期：0
• ISSN：1520-4995

文摘

Many microorganisms use flavin-dependent thymidylate synthase (FDTS) to synthesize the essential nucleotide 2′-deoxythymidine 5′-monophosphate (dTMP) from 2′-deoxyuridine 5′-monophosphate (dUMP), 5,10-methylenetetrahydrofolate (CH₂THF), and NADPH. FDTSs have a structure that is unrelated to the thymidylate synthase used by humans and a very different mechanism. Here we report nuclear magnetic resonance evidence that FDTS ionizes N3 of dUMP using an active-site arginine. The ionized form of dUMP is largely responsible for the changes in the flavin absorbance spectrum of FDTS upon dUMP binding. dUMP analogues also suggest that the phosphate of dUMP acts as the base that removes the proton from C5 of the dUMP-methylene intermediate in the FDTS-catalyzed reaction. These findings establish additional differences between the mechanisms of FDTS and human thymidylate synthase.