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., and 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. We conclude that lysine 125 plays an important role in the structure of the heparin binding region and in binding heparin with high affinity, but is not needed for the maximum heparin-induced acceleration of proteinase inhibition. We found no definitive evidence that lysines 290, 294, and 297 contribute to a heparin binding site, either as the primary site or involved in binding longer chain species.