Mapping of Allosteric Modulator Effects in the Active Site of Factor IXa.

Activated factor IX (fIXa) is a vitamin K-dependent blood coagulation serine protease involved in propagation of the coagulant response through activation of fX. Maximal enzymatic and procoagulant activity of fIXa requires the presence of several cofactors; one of which is ionic calcium, which is known to bind to a site in the protease domain of fIXa as well as several sites within the light chain Gla domain region. One of the roles of calcium appears to be allosteric modulation of the fIXa active site as evidenced by an increase in enzymatic activity towards small peptidyl substrates. We and others have additionally found that certain small hygroscopic molecules can also enhance fIXa amidolytic activity. The molecular details involved in either of these effects are not well understood. Previous studies by us have shown that a pentapeptide substrate (AGRSL; the reactive site sequence of antithrombin) is hydrolyzed by fIXa in the absence of cofactors or modulators. This hydrolysis is enhanced in the presence of ionic calcium, ethylene glycol or low molecular weight heparin suggesting effects of these molecules on the immediate active site vicinity of fIXa. In order to gain insight into the potential allosteric modulation that each of these effectors may affect in fIXa, we examined the hydrolysis of four peptide libraries based on the AGRSL pentapeptide sequence, in the presence and absence of various fIXa modulators. The four libraries synthesized were XGRSL, AXRSL, AGRXL and AGRSX; where X denotes any of the possible 20 amino acids. Each of these libraries were screened for hydrolysis by fIXa under various conditions with substrates and products being identified en masse using MALDI-TOF mass spectrometry. The results suggest that ionic calcium enhances fIXa reactivity in part by modulation of the S2 subsite in fIXa. In contrast, ethylene glycol enhances fIXa activity via modulation of the S3 subsite and heparin was found to effect the overall active site region.