title: Reaction of vascular adhesion protein-1 (VAP-1) with primary amines: mechanistic insights from isotope effects and quantitative structure-activity relationships.
abstract: Human vascular adhesion protein-1 (VAP-1) is an endothelial copper-dependent amine oxidase involved in the recruitment and extravasation of leukocytes at sites of inflammation. VAP-1 is an important therapeutic target for several pathological conditions. We expressed soluble VAP-1 in HEK293 EBNA1 cells at levels suitable for detailed mechanistic studies with model substrates. Using the model substrate benzylamine, we analyzed the steady-state kinetic parameters of VAP-1 as a function of solution pH. We found two macroscopic pK(a) values that defined a bell-shaped plot of turnover number k(cat,app) as a function of pH, representing ionizable groups in the enzyme-substrate complex. The dependence of (k(cat)/K(m))(app) on pH revealed a single pK(a) value (∼9) that we assigned to ionization of the amine group in free benzylamine substrate. A kinetic isotope effect (KIE) of 6 to 7.6 on (k(cat)/K(m))(app) over the pH range of 6 to 10 was observed with d(2)-benzylamine. Over the same pH range, the KIE on k(cat) was found to be close to unity. The unusual KIE values on (k(cat)/K(m))(app) were rationalized using a mechanistic scheme that includes the possibility of multiple isotopically sensitive steps. We also report the analysis of quantitative structure-activity relationships (QSAR) using para-substituted protiated and deuterated phenylethylamines. With phenylethylamines we observed a large KIE on k(cat,app) (8.01 ± 0.28 with phenylethylamine), indicating that C-H bond breakage is limiting for 2,4,5-trihydroxyphenylalanine quinone reduction. Poor correlations were observed between steady-state rate constants and QSAR parameters. We show the importance of combining KIE, QSAR, and structural studies to gain insight into the complexity of the VAP-1 steady-state mechanism.
authors: Manchester Interdisciplinary Biocentre, Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.
authors: Dominic P H M
authors: DP
authors: Heuts
authors: Jennet O
authors: JO
authors: Gummadova
authors: Jiayun
authors: J
authors: Pang
authors: Stephen E J
authors: SE
authors: Rigby
authors: Nigel S
authors: NS
authors: Scrutton
mesh_terms: D006631:Amine Oxidase (Copper-Containing) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001596:Benzylamines / Q000737:chemistry*; D015815:Cell Adhesion Molecules / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D013329:Structure-Activity Relationship
chemical_list: D001596:Benzylamines; D015815:Cell Adhesion Molecules; C030796:benzylamine; C076668:AOC3 protein, human; D006631:Amine Oxidase (Copper-Containing)