title: On the mechanism of dimethylarginine dimethylaminohydrolase inactivation by 4-halopyridines.
abstract: Small molecules capable of selective covalent protein modification are of significant interest for the development of biological probes and therapeutics. We recently reported that 2-methyl-4-bromopyridine is a quiescent affinity label for the nitric oxide controlling enzyme dimethylarginine dimethylaminohydrolase (DDAH) (Johnson, C. M.; Linsky, T. W.; Yoon, D. W.; Person, M. D.; Fast, W. J. Am. Chem. Soc. 2011, 133, 1553-1562). Discovery of this novel protein modifier raised the possibility that the 4-halopyridine motif may be suitable for wider application. Therefore, the inactivation mechanism of the related compound 2-hydroxymethyl-4-chloropyridine is probed here in more detail. Solution studies support an inactivation mechanism in which the active site Asp66 residue stabilizes the pyridinium form of the inactivator, which has enhanced reactivity toward the active site Cys, resulting in covalent bond formation, loss of the halide, and irreversible inactivation. A 2.18 Å resolution X-ray crystal structure of the inactivated complex elucidates the orientation of the inactivator and its covalent attachment to the active site Cys, but the structural model does not show an interaction between the inactivator and Asp66. Molecular modeling is used to investigate inactivator binding, reaction, and also a final pyridinium deprotonation step that accounts for the apparent differences between the solution-based and structural studies with respect to the role of Asp66. This work integrates multiple approaches to elucidate the inactivation mechanism of a novel 4-halopyridine "warhead," emphasizing the strategy of using pyridinium formation as a "switch" to enhance reactivity when bound to the target protein.
authors.affiliation: Division of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, Texas 78712, USA.
authors.forename: Corey M
authors.initials: CM
authors.lastname: Johnson
authors.forename: Arthur F
authors.initials: AF
authors.lastname: Monzingo
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Ke
authors.forename: Dae-Wi
authors.initials: DW
authors.lastname: Yoon
authors.forename: Thomas W
authors.initials: TW
authors.lastname: Linsky
authors.forename: Hua
authors.initials: H
authors.lastname: Guo
authors.forename: Jon D
authors.initials: JD
authors.lastname: Robertus
authors.forename: Walter
authors.initials: W
authors.lastname: Fast
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D020134:Catalytic Domain; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D054879:Halogenation; D008958:Models, Molecular; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011725:Pyridines / Q000737:chemistry* / Q000494:pharmacology*; D012996:Solutions
chemical_list: D004791:Enzyme Inhibitors; D011725:Pyridines; D012996:Solutions; D000581:Amidohydrolases; C059999:dimethylargininase; C023666:pyridine