The Role of Protein Dynamics in Thymidylate Synthase Catalysis: Variants of Conserved 2'-Deoxyuridine 5'-Monophosphate (dUMP)-Binding Tyr-261

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• 作者：Zachary Newby ; Tom T. Lee ; Richard J. Morse ; Yaoquan Liu ; Lu Liu ; Prasanna Venkatraman ; Daniel V. Santi ; Janet S. Finer-Moore ; and Robert M. Stroud
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：June 20, 2006
• 年：2006
• 卷：45
• 期：24
• 页码：7415 - 7428
• 全文大小：1164K
• 年卷期：v.45,no.24(June 20, 2006)
• ISSN：1520-4995

文摘

The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalyticmechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position showa marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, andspecificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanismof TS. However, the residue is part of the structural core of closed ternary complexes of TS, andconservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values ofk_cat/K_m. Moderate increases in K_m values for both the substrate and cofactor upon mutation of Tyr-261arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine.Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme andin shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystalsof a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop ink_cat/K_m. These results underline the important contribution of dynamics to catalysis in TS.