An Essential Role for Water in an Enzyme Reaction Mechanism: The Crystal Structure of the Thymidylate Synthase Mutant E58Q

详细信息    查看全文

• 作者：Carleton R. Sage ; Earl E. Rutenber ; Thomas J. Stout ; and Robert M. Stroud
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：December 17, 1996
• 年：1996
• 卷：35
• 期：50
• 页码：16270 - 16281
• 全文大小：599K
• 年卷期：v.35,no.50(December 17, 1996)
• ISSN：1520-4995

文摘

A water-mediated hydrogen bond network coordinated byglutamate 60(58) appears to playan important role in the thymidylate synthase (TS) reaction mechanism.We have addressed the role ofglutamate 60(58) in the TS reaction by cocrystallizing theEscherichia coli TS mutant E60(58)Q withdUMP and the cofactor analog CB3717 and have determined the X-raycrystal structure to 2.5 Å resolutionwith a final R factor of 15.2%(R_free = 24.0%). Using differenceFourier analysis, we analyzed directlythe changes that occur between the wild-type and mutant structures.The structure of the mutant enzymesuggests that E60(58) is not required to properly position theligands in the active site and that thecoordinated hydrogen bond network has been disrupted in the mutant,providing an atomic resolutionexplanation for the impairment of the TS reaction by the E60(58)Qmutant and confirming the proposalthat E60(58) coordinates this conserved hydrogen bond network.The structure also provides insight intothe role of specific waters in the active site which have beensuggested to be important in the TS reaction.Finally, the structure shows a unique conformation for thecofactor analog, CB3717, which has implicationsfor structure-based drug design and sheds light on the controversysurrounding the previously observedenzymatic nonidentity between the chemically identical monomers of theTS dimer.