Coagulation enzymes are trypsin-like serine proteases with a PI specificity of Arg, but unlike trypsin, they exhibit high degrees of primary substrate specificity, require complex formation with a cofactor to function and are inhibited by a small subset of protease inhibitors. Although the three dimensional structure of these enzymes are highly conserved, certain residues on the surface, particularly those found in the extended binding pockets vary among coagulation proteases. We hypothesize that the variable residues, at least in part, determine the differences in primary substrate specificity in coagulation enzymes. We will test this hypothesis by mutagenesis of the variant residues in protein C, factor X and prothrombin. The experimental strategy we will use is to identify the variant residue(s) by homology modeling, replace these residue(s) with those found in the analogous site of a different family member and determine if the specificity is switched toward that of the donor enzyme. We further hypothesize that cofactors may influence the substrate specificity by altering the conformation of these-variant residue(s) by allosteric mechanisms and eliminating potentially inhibitory interactions with substrates. This hypothesis will be tested using both wild type or mutant enzymes containing specific-mutations at the S3-S3' sites and wild type or mutant substrates containing complementary mutations at the P3-P3' sites. The rate of activation of wild type substrates by mutant enzymes or mutant substrates by wild type enzymes in the presence and absence of cofactors will be compared. If the hypothesis is correct, then the mutagenesis should at least partially eliminate the cofactor requirement for zymogen activation. The results will be extended by analysis of inhibition profiles using mutants of bovine pancreatic trypsin inhibitor (BPTI) in which the P3-P3' residues of the inhibitor are substituted with those of the wild type or mutant substrates. In this case a lower Ki with or.without a cofactor present will support the hypothesis. The study will expand our knowledge of the molecular basis of coagulation factor specificity and the mechanism of cofactor function.
Showing the most recent 10 out of 47 publications
