The Role of Factor XIa (FXIa) Catalytic Domain Exosite Residues in Substrate Catalysis and Inhibition by the Kunitz Protease Inhibitor Domain of Protease Nexin 2

Ya-Chi Su,Tara N Miller,Duraiswamy Navaneetham,Robert T Schoonmaker,Dipali Sinha,Peter N Walsh

doi:10.1074/jbc.m111.257527

Abstract

To select residues in coagulation factor XIa (FXIa) potentially important for substrate and inhibitor interactions, we examined the crystal structure of the complex between the catalytic domain of FXIa and the Kunitz protease inhibitor (KPI) domain of a physiologically relevant FXIa inhibitor, protease nexin 2 (PN2). Six FXIa catalytic domain residues (Glu(98), Tyr(143), Ile(151), Arg(3704), Lys(192), and Tyr(5901)) were subjected to mutational analysis to investigate the molecular interactions between FXIa and the small synthetic substrate (S-2366), the macromolecular substrate (factor IX (FIX)) and inhibitor PN2KPI. Analysis of all six Ala mutants demonstrated normal K(m) values for S-2366 hydrolysis, indicating normal substrate binding compared with plasma FXIa; however, all except E98A and K192A had impaired values of k(cat) for S-2366 hydrolysis. All six Ala mutants displayed deficient k(cat) values for FIX hydrolysis, and all were inhibited by PN2KPI with normal values of K(i) except for K192A, and Y5901A, which displayed increased values of K(i). The integrity of the S1 binding site residue, Asp(189), utilizing p-aminobenzamidine, was intact for all FXIa mutants. Thus, whereas all six residues are essential for catalysis of the macromolecular substrate (FIX), only four (Tyr(143), Ile(151), Arg(3704), and Tyr(5901)) are important for S-2366 hydrolysis; Glu(98) and Lys(192) are essential for FIX but not S-2366 hydrolysis; and Lys(192) and Tyr(5901) are required for both inhibitor and macromolecular substrate interactions.

Highlights

IntroductionWe have utilized this structural information (Fig. 1) to examine the architecture of residues in close proximity to the catalytic triad and to select a number of residues within the catalytic domain of factor XIa (FXIa) (Asp, Lys192, Ser195, Asp189, Gly193, Tyr143, Ile151, Arg3704, and Tyr5901) that make
The isolated Kunitz-type protease inhibitor (KPI) domain and the factor XIa (FXIa) catalytic domain have been co-crystallized, and their structure has been solved to a resolution of 2.6 Å [21]. This structure combined with a mutational analysis of the KPI domain has been used to identify a number of residues within two loop structures (Loop 1 and Loop 2) within the KPI domain postulated to interact with corresponding residues within the catalytic domain of FXIa that are potentially important for both inhibitor and substrate interactions
The mechanism by which serine proteases recognize and cleave their substrates and inhibitors involves the formation of a catalytic triad (His57, Asp102, and Ser195), located at the entrance of the substrate/inhibitor-binding pocket, the geometry of which is stabilized by hydrogen bonds [34]

Summary

Introduction

We have utilized this structural information (Fig. 1) to examine the architecture of residues in close proximity to the catalytic triad and to select a number of residues within the catalytic domain of FXIa (Asp, Lys192, Ser195, Asp189, Gly193, Tyr143, Ile151, Arg3704, and Tyr5901) that make. We have made selected mutations at these identified exosite residues (i.e. excluding the active site), and examined the resulting enzymes (after activation to FXIa) in the hydrolysis of the peptide substrate S-2366, in the activation of the macromolecular substrate, FIX, and in the regulation of FXIa by PN2. Whereas mutations at four of these sites (Glu, Tyr143, Ile151, and Arg3704) resulted in normal values of Ki for inhibition by PN2KPI, in contrast, mutations at the other two sites (Lys192 and Tyr5901) resulted in enzymes with impaired interactions with PN2KPI as well as macromolecular substrate catalysis

Methods

Results

Conclusion