title: The oligosaccharide side chain on Asn-135 of alpha-antithrombin, absent in beta-antithrombin, decreases the heparin affinity of the inhibitor by affecting the heparin-induced conformational change.
abstract: The beta-form of antithrombin, lacking a carbohydrate side chain on Asn-135, is known to bind heparin more tightly than the fully glycosylated alpha-form. The molecular basis for this difference in affinity was elucidated by rapid-kinetic studies of the binding of heparin and the antithrombin-binding heparin pentasaccharide to plasma and recombinant forms of alpha- and beta-antithrombin. The dissociation equilibrium constant for the first step of the two-step mechanism of binding of both heparin and pentasaccharide to alpha-antithrombin was only slightly higher than that for the binding to the beta-form. The oligosaccharide at Asn-135 thus at most moderately interferes with the initial, weak binding of heparin to alpha-antithrombin. In contrast, the rate constant for the conformational change induced by heparin and pentasaccharide in the second binding step was substantially lower for alpha-antithrombin than for beta-antithrombin. Moreover, the rate constant for the reversal of this conformational change was appreciably higher for the alpha-form than for the beta-form. The carbohydrate side chain at Asn-135 thus reduces the heparin affinity of alpha-antithrombin primarily by interfering with the heparin-induced conformational change. These and previous results suggest a model in which the Asn-135 oligosaccharide of alpha-antithrombin is oriented away from the heparin binding site and does not interfere with the first step of heparin binding. This initial binding induces conformational changes involving extension of helix D into the adjacent region containing Asn-135, which are transmitted to the reactive-bond loop. The resulting decreased conformational flexibility of the Asn-135 oligosaccharide and its close vicinity to the heparin binding site destabilize the activated relative to the native conformation. This effect results in a higher energy for inducing the activated conformation in alpha-antithrombin, leading to a decrease in heparin binding affinity.
authors.affiliation: Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala Biomedical Center.
authors.forename: B
authors.initials: B
authors.lastname: Turk
authors.forename: I
authors.initials: I
authors.lastname: Brieditis
authors.forename: S C
authors.initials: SC
authors.lastname: Bock
authors.forename: S T
authors.initials: ST
authors.lastname: Olson
authors.forename: I
authors.initials: I
authors.lastname: Björk
mesh_terms: D000990:Antithrombin III / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001216:Asparagine / Q000737:chemistry*; D001665:Binding Sites; D065427:Factor Xa Inhibitors; D006031:Glycosylation; D006493:Heparin / Q000008:administration & dosage / Q000378:metabolism* / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D009844:Oligosaccharides / Q000737:chemistry*; D011487:Protein Conformation / Q000187:drug effects*; D013329:Structure-Activity Relationship; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D065427:Factor Xa Inhibitors; D009844:Oligosaccharides; D001216:Asparagine; D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin