Structural and Functional Significance of Amino Acid Lysine192 (chymotrypsin numbering) in Factor XIa and Factor VIIa

In blood coagulation serine proteases, amino acid 192 (chymotrypsin numbering) influences substrate and inhibitor specificity, and also forms a peptide bond with Gly 193 , a critical part of the protease oxyanion hole. Residue 192 is either Glu or Gln in several serine proteases including thrombin, factor (F) Xa, FIXa, and activated protein C. However, it is Lys in factor FXIa and FVIIa. Both FXIa and FVIIa/tissue factor (TF) share FIX as their substrate. In addition, FVIIa/TF activates FX and is inhibited by TF pathway inhibitor (TFPI), whereas FXIa is inhibited by diisopropylfluorophosphate (DFP), p -aminobenzamidine ( p- AB), antithrombin and amyloid-b precursor protein Kunitz domain (AbPP). We investigated the importance of Lys 192 in FXIa and FVIIa for substrate/inhibitor specificity and oxyanion hole formation. Recombinant FXIa was prepared with Pro (FXIa K192P ), Glu (FXIa K192E ) or Gln (FXIa K192Q ) substitutions for Lys 192 . Rate constants for inhibition by DFP were similar for wild type FXIa (FXIa WT ), FXIa K192E and FXIa K192Q , but 80-fold slower for FXIa K192P . Only FXIa K192P differed in binding to p AB compared to FXIa WT . These data indicate that Pro 192 causes distortion of the oxyanion hole and S1 binding site. All mutants bound the FXIa substrate FIX normally. However, catalysis of FIX was impaired 400, 35, and 110-fold for FXIa K192P , FXIa K192E , and FXIa K192Q , respectively, while k 2 for inhibition by antithrombin was reduced 160, 15 and 16-fold. FXIa K192P bound to AbPP with 800-fold decreased affinity; while FXIa K192E and FXIa K192Q bound with 6-10-fold higher affinity. Modeling studies indicate that there is loss of the normal interaction between Lys 192 and the substrate/inhibitor P3’ residue in interactions between FXIa K192E and FXIa K192Q and factor IX and antithrombin. The K i for Glu and Gln mutant inhibition by AbPP is slightly better probably due to the ability of these two amino acids to make two H-bonds as compared to only one for Lys 192 . Lys 192 makes H-bond with the carbonyl O of Gly 12 in AbPP, whereas Glu and Gln at this position make H-bonds with the carbonyl O of Cys 14 and Ala 16 in AbPP. In FXIa K192P , the oxyanion hole is impaired due to a flip in the 192-193 peptide bond causing the amide N of Gly 193 to point away from the oxyanion hole, resulting in a severe impairment of all catalytic functions. Thus a non-proline residue 192 is essential for proper oxyanion hole formation in serine proteases, and Lys 192 in FXIa contributes significantly towards macromolecular substrate catalysis and inhibitor binding. In contrast to FXIa WT , the 192-193 peptide bond in FVIIa WT ± TF is already in a nonstandard conformation in which the amide N of Gly 193 points away, and the carbonyl O of the Lys 192 points into the oxyanion hole (Bajaj, et al., J. Biol. Chem.281, 24873-24888, 2006). The H-bond between the amide N of Gly 193 and the carbonyl side chain of Gln 143 , a residue unique to FVIIa, maintains this nonstandard conformation in FVIIa. Notably, upon substrate binding the 192-193 bond flips 180° such that the oxyanion hole is fully formed. Now the H-bond is formed between the amide N of Gly 193 and the carbonyl oxygen of the transition state intermediate. For this to occur, the H-bond between the amide N of Gly 193 and the carbonyl side chain of Gln 143 must first be broken. Previous studies have shown that changing Lys 192 to Glu or Gln in FVIIa severely impairs its biologic activity. However, the structural basis for this impairment is not understood. We performed molecular modeling studies which reveal that the side chain of the Glu 192 carboxyl group or of the Gln 192 carbonyl group makes strong H-bond with the side chain NH 2 group of Gln 143 ; this is not possible for Lys 192 . Thus in Glu 192 or Gln 192 mutant of FVIIa, it is energetically difficult to break the H-bond between the amide N of Gly 193 and the carbonyl side chain of Gln 143 because the H-bond involving the side chain of Glu 192 or Gln 192 and the Gln 143 must be simultaneously broken. This provides a structural rational for the impaired function of FVIIa K192E or FVIIa K192Q . To test this concept further, we prepared FVIIa in which Lys 192 was replaced with alanine. All properties of this alanine mutant were normal substantiating the proposed structural role of Lys 192 in FVIIa. This structural role is unique to FVIIa among all known serine proteases, as is the presence of residue Gln 143 that contributes to this phenomenon.