- Department of Molecular Biology, Swedish University of Agricultural Sciences
Zhu, Tianqing; Ubhayasekera, Wimal; Nickolaus, Noelle; Sun, Wei; Tingsborg, Susanne; Mowbray, Sherry; Schedin-Weiss, Sophia
Zn(2+) ions were found to efficiently inhibit activated protein C (APC), suggesting a potential regulatory function for such inhibition. APC activity assays employing a chromogenic peptide substrate demonstrated that the inhibition was reversible and the apparent K(1) was 13 +/- 2 mu M. k(cat) was seven fold decreased whereas K(M) was unaffected in the presence of 10 mu M Zn(2+). The inhibitory effect of Zn(2+) on APC activity was also observed when factor Va was used as a substrate in an assay coupled to a prothrombinase assay. The interaction of Zn(2+) with APC was accompanied by a reversible similar to 40% decrease in tryptophan fluorescence, consistent with the ion inducing a conformational change in the protein. The apparent K(D) was 7.4 +/- 1.5 mu M and thus correlated well with the apparent K(1). In the presence of physiological Ca(2+) concentration the K(1) and K(D) values were three to four fold enhanced, presumably due to the Ca(2+)-induced conformational change affecting the conformation of the Zn(2+)-binding site. The inhibition mechanism was noncompetitive both in the absence and presence of Ca(2+). Comparisons of sequences and structures suggested several possible sites for zinc binding. The magnitude of the apparent K(1) in relation to the blood and platelet concentrations of Zn(2+) supports a physiological role for this ion in the regulation of anticoagulant activity of APC. These findings broaden the understanding of this versatile serine protease and enable the future development of potentially more efficient anticoagulant APC variants for treatments of thrombotic diseases.
Activated protein C; serine protease; zinc; blood coagulation; enzyme inhibition
Thrombosis and Haemostasis
2010, Volume: 104, number: 3, pages: 544-553
Publisher: SCHATTAUER GMBH-VERLAG MEDIZIN NATURWISSENSCHAFTEN