Reaction of Vascular Adhesion Protein-1 (VAP-1) with Primary Amines

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 (ϳ9) 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.Copper amine oxidases or semicarbazide-sensitive amine oxidases (CAOs/SSAOs 3 ; EC 1.4.3.21/EC 1.4.3.22) comprise a group of copper-dependent enzymes that catalyze the oxidative deamination of primary amines to the corresponding aldehydes with concomitant release of hydrogen peroxide and ammonia. (R is a phenyl moiety in case of benzylamine and a benzyl moiety in case of phenylethylamine.)From the viewpoint of structure and mechanism, copper-dependent amine oxidases have been researched extensively for several decades, but relatively little is known about their physiological function.Human vascular adhesion protein-1 (VAP-1) is a dimeric membrane bound and circulating protein that is encoded by the AOC3 gene and is expressed mainly in endothelial cells of blood vessels, adipocytes, and smooth muscle cells (1-3). VAP-1 is a protein comprising two structural domains: an adhesive domain that targets leukocytes for transmigration and an amine oxidase domain (2, 4). At sites of inflammation on the endothelial cell surface, VAP-1 is involved in the recruitment and extravasation of lymphocytes and neutrophils. VAP-1 activity generates important signaling compounds such as hydrogen peroxide (5, 6). SSAO activity is also involved in glucose transport in adipose cells (1...

Human copper-dependent amine oxidases

“…Recombinant forms of AOC3 (rAOC3) have been produced in Chinese hamster ovary (CHO) cells (46,60), Ax endothelial cells (67), HEK293 cells (68,69), and Drosophila S 2 cells (70). Recombinant AOC3s produced in HEK and S2 cells were expressed without the transmembrane domain (i.e.…”

“…Recently, two independent detailed biochemical studies were conducted on a rAOC3 produced in HEK293-EBNA1 cells and insect cells (68,70). These rAOC3s were purified to >95% homogeneity from serum-free media and were detected as a single band at ~ 100 kDa (68,70).…”

“…These rAOC3s were purified to >95% homogeneity from serum-free media and were detected as a single band at ~ 100 kDa (68,70). The stoichiometric amount of titratable TPQ cofactor was ~19% and ~6%, respectively, for the rAOC3s produced in HEK cells and insect cells.…”

“…The pH-dependency curve of the steady-state kinetic parameters of rAOC3 produced in HEK cells was fit using nonlinear regression (68). A bell-shaped curve was fit to the apparent k cat versus pH plot, with two macroscopic p K a values (7.0 ± 0.2 and 10.0 ± 0.4) representing ionizable groups in the rAOC3-substrate complex.…”

Human lysyl oxidase-like 2

“…Titration with phenylhydrazine revealed only ~50% formation of the LTQ cofactor in native LOX isolated from bovine calf aorta (32). Also, in recombinant CAOs from yeast and human cells only ~20% TPQ (another tyrosine-derived quinone cofactor) formation was detected (35,36). Moreover, neither recombinant D. melanogaster lysyl oxidase-like 1 (rDmLOXL-1) produced in S2 cells nor rLOXL2 produced in E. coli contains LTQ upon initial isolation.…”