Prioritization of Charge over Geometry in Transition State Analogues of a Dual Specificity Protein Kinase

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• 作者：Liu Xiaoxia ; James P. Marston ; Nicola J. Baxter ; Andrea M. Hounslow ; Zhao Yufen ; G. Michael Blackburn ; Matthew J. Cliff ; Jonathan P. Waltho
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：March 23, 2011
• 年：2011
• 卷：133
• 期：11
• 页码：3989-3994
• 全文大小：868K
• 年卷期：v.133,no.11(March 23, 2011)
• ISSN：1520-5126

文摘

The direct observation of a transition state analogue (TSA) complex for tyrosine phosphorylation by a signaling kinase has been achieved using ¹⁹F NMR analysis of MEK6 in complex with tetrafluoroaluminate (AlF₄^{鈭?/sup>), ADP, and p38伪 MAP kinase (acceptor residue: Tyr182). Solvent-induced isotope shifts and chemical shifts for the AlF₄鈭?/sup> moiety indicate that two fluorine atoms are coordinated by the two catalytic magnesium ions of the kinase active site, while the two remaining fluorides are liganded by protein residues only. An equivalent, yet distinct, AlF₄鈭?/sup> complex involving the alternative acceptor residue in p38伪 (Thr180) is only observed when the Tyr182 is mutated to phenylalanine. The formation of octahedral AlF₄鈭?/sup> species for both acceptor residues, rather than the trigonal bipyramidal AlF₃0 previously identified in the only other metal fluoride complex with a protein kinase, shows the requirement of MEK6 for a TSA that is isoelectronic with the migrating phosphoryl group. This requirement has hitherto only been demonstrated for proteins having a single catalytic magnesium ion.}