Correlation of factor IXa subsite modulations with effects on substrate discrimination.
BACKGROUND
A key feature of factor IXa is its allosteric transformation from an enzymatically latent form into a potent procoagulant. Although several small molecules have been found to be capable of partially affecting FIXa function (i.e. ethylene glycol, Ca(2+), and low molecular weight heparin [LMWH]), the resulting modest changes in peptidolytic activity have made the study of their mechanisms of action challenging. As these effects provide hints about potential regulatory forces that may be operational in the full expression of FIXa coagulant activity, their description remains of great interest. Studies of crystal structures have yielded insights into the structural changes induced by these effectors, but there remains a paucity of information to correlate any given structural change with specific consequences for FIXa function.


OBJECTIVE
To correlate structural changes induced by these modulators with defined consequences for FIXa substrate discrimination and function.


METHODS
A peptidomics-based mass spectrometry (MS) approach was used to examine the patterns of hydrolysis of four combinatorial chemistry-derived pentapeptide libraries by FIXa under various conditions in a soluble, active enzyme system.


RESULTS
Ethylene glycol specifically altered the S3 subsite of FIXa to render it more tolerant to side chains at the P3 substrate position, whereas Ca(2+) enhanced tolerance at the S2 subsite. In contrast, LMWH altered both the S2 and S1' subsites.


CONCLUSIONS
These results demonstrate the role of plasticity in regulating FIXa function with respect to discrimination of extended substrate sequences, as well as providing crucial insights into active site modulations that may be capitalized on by various physiologic cofactors of FIXa and in future drug design.
Center for Biomedical Research, The University of Texas Health Science Center at Tyler, Tyler, TX 75708, USA. pierre.neuenschwander@uthct.edu
P F
PF
Neuenschwander
K J
KJ
Deadmond
K
K
Zepeda
J
J
Rutland
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D020134:Catalytic Domain; D002863:Chromogenic Compounds / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques; D019855:Ethylene Glycol / Q000378:metabolism; D015949:Factor IXa / Q000737:chemistry / Q000378:metabolism*; D006495:Heparin, Low-Molecular-Weight / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D009842:Oligopeptides / Q000378:metabolism; D019151:Peptide Library; D057786:Peptidomimetics / Q000378:metabolism; D011487:Protein Conformation; D040901:Proteomics / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D002863:Chromogenic Compounds; D006495:Heparin, Low-Molecular-Weight; D009842:Oligopeptides; D019151:Peptide Library; D057786:Peptidomimetics; D015949:Factor IXa; D019855:Ethylene Glycol; D002118:Calcium