Structural and Functional Significance of Amino Acid Lysine192 (chymotrypsin numbering) in Factor XIa and Factor VIIa.
Authors: Schmidt Amy E , Agah Sayeh , Sun Mao-fu , Padmanabhan Kaillathe , Cascio Duilio , Gailani David , Bajaj S. Paul 
Published: April 2020

Abstract
In blood coagulation serine proteases, amino acid 192 (chymotrypsin numbering) influences substrate and inhibitor specificity, and also forms a peptide bond with Gly193, 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 Lys192 in FXIa and FVIIa for substrate/inhibitor specificity and oxyanion hole formation. Recombinant FXIa was prepared with Pro (FXIaK192P), Glu (FXIaK192E) or Gln (FXIaK192Q) substitutions for Lys192. Rate constants for inhibition by DFP were similar for wild type FXIa (FXIaWT), FXIaK192E and FXIaK192Q, but 80-fold slower for FXIaK192P. Only FXIaK192P differed in binding to pAB compared to FXIaWT. These data indicate that Pro192 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 FXIaK192P, FXIaK192E, and FXIaK192Q, respectively, while k2 for inhibition by antithrombin was reduced 160, 15 and 16-fold. FXIaK192P bound to AbPP with 800-fold decreased affinity; while FXIaK192E and FXIaK192Q bound with 6-10-fold higher affinity. Modeling studies indicate that there is loss of the normal interaction between Lys192 and the substrate/inhibitor P3’ residue in interactions between FXIaK192E and FXIaK192Q and factor IX and antithrombin. The Ki 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 Lys192. Lys192 makes H-bond with the carbonyl O of Gly12 in AbPP, whereas Glu and Gln at this position make H-bonds with the carbonyl O of Cys14 and Ala16 in AbPP. In FXIaK192P, the oxyanion hole is impaired due to a flip in the 192-193 peptide bond causing the amide N of Gly193 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 Lys192 in FXIa contributes significantly towards macromolecular substrate catalysis and inhibitor binding. In contrast to FXIaWT, the 192-193 peptide bond in FVIIaWT ± TF is already in a nonstandard conformation in which the amide N of Gly193 points away, and the carbonyl O of the Lys192 points into the oxyanion hole (Bajaj, et al., J. Biol. Chem.281, 24873-24888, 2006). The H-bond between the amide N of Gly193 and the carbonyl side chain of Gln143, 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 Gly193 and the carbonyl oxygen of the transition state intermediate. For this to occur, the H-bond between the amide N of Gly193 and the carbonyl side chain of Gln143 must first be broken. Previous studies have shown that changing Lys192 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 Glu192 carboxyl group or of the Gln192 carbonyl group makes strong H-bond with the side chain NH2 group of Gln143; this is not possible for Lys192. Thus in Glu192 or Gln192 mutant of FVIIa, it is energetically difficult to break the H-bond between the amide N of Gly193 and the carbonyl side chain of Gln143 because the H-bond involving the side chain of Glu192 or Gln192 and the Gln143 must be simultaneously broken. This provides a structural rational for the impaired function of FVIIaK192E or FVIIaK192Q. To test this concept further, we prepared FVIIa in which Lys192 was replaced with alanine. All properties of this alanine mutant were normal substantiating the proposed structural role of Lys192 in FVIIa. This structural role is unique to FVIIa among all known serine proteases, as is the presence of residue Gln143 that contributes to this phenomenon.