Lysine-heparin interactions in antithrombin. Properties of K125M and K290M,K294M,K297M variants.
Lysine residues in two different regions of antithrombin have been proposed to be involved in heparin binding and heparin-mediated acceleration of proteinase inhibition. Lysine 125 has been implicated as an essential heparin binding residue from chemical modification studies [Peterson, C. B., Noyes, C. M., Pecon, J. M., Church, F. C., & Blackburn, M. N. (1987) J. Biol. Chem. 262, 8061-8065] whereas lysines 290, 294, and 297 have been proposed from model building studies to constitute the heparin binding site [Villanueva, G. B. (1984) J. Biol. Chem. 259, 2531-2536]. To evaluate both of these proposals, we have prepared two variant human antithrombins, K125M and K290M,K294M,K297M, in which these lysines have been changed by site-directed mutagenesis to methionines. The K290M,K294M,K297M variant had properties very similar to those of wild-type recombinant antithrombin in affinity for heparin, and in rates of inhibition of thrombin and factor Xa. In contrast, K125M antithrombin had reduced affinity for both heparin pentasaccharide and full-length heparin, corresponding to delta delta Gs of 3.1 and 2.0 kcal mol-1, respectively. However, this variant was still able to inhibit both thrombin and factor Xa. Whereas the rate of thrombin inhibition was similar to that of wild-type antithrombin, the rate of factor Xa inhibition was enhanced between 2- and 3-fold, suggesting a role for lysine 125 in the allosteric coupling between the heparin binding site and the reactive center region. At saturation with either heparin pentasaccharide or full-length high-affinity heparin, the rates of inhibition of both proteinases were similar to those of wild-type antithrombin for both the K125M and K290M,K294M,K297M variants.(ABSTRACT TRUNCATED AT 250 WORDS)
Fan
B
B
Department of Biochemistry, University of Illinois-Chicago 60612.
Turko
I V
IV
Gettins
P G
PG
D000991:Antithrombins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D065427:Factor Xa Inhibitors; D006493:Heparin / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008715:Methionine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D013917:Thrombin / Q000037:antagonists & inhibitors
D000991:Antithrombins; D065427:Factor Xa Inhibitors; D009844:Oligosaccharides; D011994:Recombinant Proteins; C049714:IC 831423; D006493:Heparin; D008715:Methionine; D013917:Thrombin; D008239:Lysine