Discovery of a sensitive, selective, and tightly binding fluorogenic substrate of bovine plasma amine oxidase.
We report a novel fluorogenic substrate of bovine plasma amine oxidase (BPAO), namely, (2-(6-(aminomethyl)naphthalen-2-yloxy)ethyl)trimethylammonium (ANETA), which displays extremely tight binding to BPAO (K(m) 183 +/- 14 nM) and yet is metabolized fairly quickly (k(cat) 0.690 +/- 0.010 s(-1)), with the aldehyde turnover product (2-(6-formylnaphthalen-2-yloxy)ethyl)trimethylammonium serving as a real time reporting fluorophore of the enzyme activity. This allowed for the development of a fluorometric noncoupled assay that is 2 orders of magnitude more sensitive than the spectrophotometric benzylamine assay. The discovery of ANETA involved elaboration of the lead compound 6-methoxy-2-naphthalenemethaneamine by structure-based design, which recognized the ancillary cation binding site of BPAO as the most significant structural features controlling binding affinity. Structure-based design further ensured a high level of selectivity: ANETA is a good substrate of BPAO but is not a substrate of either porcine kidney diamine oxidase (pkDAO) or rat liver monoamine oxidase (MAO-B). ANETA represents the first highly sensitive, selective, and tight binding fluorogenic substrate of a copper amine oxidase that is able to respond directly to the enzyme activity in real time.
Ling
Ke-Qing
KQ
Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA. kqling@hotmail.com
Sayre
Lawrence M
LM
D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D008725:Bis-Trimethylammonium Compounds / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D003198:Computer Simulation; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D006860:Hydrogen Bonding; D008956:Models, Chemical; D015394:Molecular Structure; D009281:Naphthalenes / Q000737:chemistry* / Q000378:metabolism*; D010949:Plasma / Q000201:enzymology; D012680:Sensitivity and Specificity; D013237:Stereoisomerism; D013329:Structure-Activity Relationship
C534893:(2-(6-(aminomethyl)naphthalen-2-yloxy)ethyl)trimethylammonium; D008725:Bis-Trimethylammonium Compounds; D005456:Fluorescent Dyes; D009281:Naphthalenes; D006631:Amine Oxidase (Copper-Containing)