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)