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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.

SUBMITTER: Heuts DP

PROVIDER: S-EPMC3190999 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Reaction of vascular adhesion protein-1 (VAP-1) with primary amines: mechanistic insights from isotope effects and quantitative structure-activity relationships.

Heuts Dominic P H M DP Gummadova Jennet O JO Pang Jiayun J Rigby Stephen E J SE Scrutton Nigel S NS

The Journal of biological chemistry 20110707 34

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 ...[more]

PMID: 21737458

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Dataset Information

Reaction of vascular adhesion protein-1 (VAP-1) with primary amines: mechanistic insights from isotope effects and quantitative structure-activity relationships.

Publications

Reaction of vascular adhesion protein-1 (VAP-1) with primary amines: mechanistic insights from isotope effects and quantitative structure-activity relationships.

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