期刊
CHEMBIOCHEM
卷 18, 期 15, 页码 1551-1556出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201700104
关键词
covalent inhibitors; covalent probes; enzyme inactivation; halopyridines
资金
- National Institutes of Health (NIH) [R01 GM069754]
- Robert A. Welch Foundation [F-1572]
- NIH [1K12 GM102745]
We have investigated 4-halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chemical probes. Nonenzymatic reactivity of 4-chloropyridine with amino acids and thiols was ranked with respect to common covalent protein-modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse, fragment-sized 4-halopyridines inactivated human dimethylarginine dimethylaminohydrolase-1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off-pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2-fluoromethyl-substituted 4-chloropyridines demonstrated that the pK(a) and k(inact)/K-I values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysis-dependent. 4-Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.
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