Study of the Phosphoryl‐Transfer Mechanism of Shikimate Kinase by NMR Spectroscopy. Issue 8 (21st January 2016)

• Record Type: Journal Article
• Authors: Prado, Verónica; Lence, Emilio; Vallejo, Juan A.; Beceiro, Alejandro; Thompson, Paul; Hawkins, Alastair R.; González‐Bello, Concepción
• Is Part Of: Chemistry. Volume 22:Issue 8(2016)
• Page Start: 2758
• Page End: 2768
• Publication Date: 2016-01-21
• Subjects: drug discovery -- enzyme catalysis -- molecular dynamics -- molecular modelling -- reaction mechanism; Chemistry -- Periodicals; 540
• DOI: 10.1002/chem.201504438
• Languages: English
• ISSNs: 0947-6539

Abstract

Abstract: The phosphoryl‐transfer mechanism of shikimate kinase from Mycobacterium tuberculosis and Helicobacter pylori, which is an attractive target for antibiotic drug discovery, has been studied by 1D 1 H and 31 P NMR spectroscopy. Metaphosphoric acid proved to be a good mimetic of the metaphosphate intermediate and facilitated the ready and rapid evaluation by NMR spectroscopic analysis of a dissociative mechanism. The required closed form of the active site for catalysis was achieved by the use of ADP (product) or two synthetic ADP analogues (AMPNP, AMPCP). Molecular dynamics simulation studies reported here also revealed that the essential arginine (Arg116/Arg117 in H. pylori and M. tuberculosis, respectively), which activates the γ‐phosphate group of ATP for catalysis and triggers the release of the product for turnover, would also be involved in the stabilisation of the metaphosphate intermediate during catalysis. We believe that the studies reported here will be helpful for future structure‐based design of inhibitors of this attractive target. The approach is also expected be useful for studies on the possible dissociative mechanism of other kinase enzymes. Abstract : A model intermediate : An NMR approach to study the possible dissociative phosphoryl‐transfer mechanism of shikimate kinase is reported (see scheme). Metaphosphoric acid proved to be a good mimetic of the reaction intermediate. A closed conformation of the active site, which is required for catalysis, was achieved by using ADP or synthetic analogues of ADP (AMPNP, AMPCP).