scholarly journals Mechanisms and specificity of factor XIa and trypsin inhibition by protease nexin 2 and basic pancreatic trypsin inhibitor

2010 ◽  
Vol 148 (4) ◽  
pp. 467-479 ◽  
Author(s):  
Duraiswamy Navaneetham ◽  
Dipali Sinha ◽  
Peter N. Walsh
Keyword(s):  
Trypsin Inhibitor ◽  
Trypsin Inhibition ◽  
Basic Pancreatic Trypsin Inhibitor ◽  
Factor Xia ◽  
Pancreatic Trypsin Inhibitor ◽  
Protease Nexin

The Role of the P1 Residue of Protease Nexin 2 and Basic Pancreatic Trypsin Inhibitor in the Mechanism of Factor XIa Inhibition.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2015-2015
Author(s):  
Duraiswamy Navaneetham ◽  
Dipali Sinha ◽  
Peter N. Walsh
Keyword(s):  
Steady State ◽  
Protease Inhibitor ◽  
Trypsin Inhibitor ◽  
Tight Binding ◽  
Basic Pancreatic Trypsin Inhibitor ◽  
Factor Xia ◽  
Pancreatic Trypsin Inhibitor ◽  
Kunitz Type ◽  
Protease Nexin ◽  
Kunitz Type Protease Inhibitor

Abstract Factor XIa (FXIa), a plasma serine protease that activates FIX, is regulated by protease nexin 2 (PN2), a Kunitz-type protease inhibitor (KPI) secreted on platelet activation. The Kunitz protease inhibitor domain of protease nexin 2 (PN2KPI) is highly homologous (45% primary sequence identity) to basic pancreatic trypsin inhibitor (BPTI, another Kunitz-type protease inhibitor) and their backbone tertiary structures are nearly identical (J Biol Chem280: 36165, 2005; J Mol Biol230: 919, 2005). However, PN2KPI (Ki 1 nM) is 600-fold more potent as a FXIa inhibitor than bovine basic pancreatic trypsin inhibitor (BPTI; Ki 630 nM). The present study is aimed at examining why these two structurally similar inhibitors are so different in their affinities and specificities in FXIa inhibition and analyzing the mechanisms of FXIa inhibition by these two inhibitors. Reasoning that the P1 residue (Arg15 in PN2KPI and Lys15 in BPTI) might play a crucial role in determining the affinity and specificity for FXIa we expressed BPTI-K15R (mimicking PN2KPI at the P1 site) and PN2KPI-R15K (mimicking BPTI at the P1 site) and examined their inhibitory properties. BPTI-K15R was found to inhibit FXIa with a Ki (10 nM) ~60-fold tighter than BPTI (Ki 630 nM), whereas PN2KPI-R15K inhibited FXIa with a Ki (32 nM) that was 32- fold impaired compared with PN2KPI (Ki 1 nM). Progress curves of peptidyl fluorogenic substrate (Boc-Glu-Ala-Arg-AMC) hydrolysis by FXIa in the presence of all the inhibitors except BPTI clearly demonstrated higher initial rates that continuously decreased until reaching a steady state, typical of slow inhibition. Progress curves obtained using BPTI, on the other hand, were found to be linear at all concentrations of the inhibitor. Analysis using steady state kinetics clearly demonstrated that BPTI is a competitive inhibitor in FXIa hydrolysis of the peptidyl chromogenic substrate, pyro-Glu-Pro-Arg-pNA. Analysis of progress curves using kinetic equations for slow, tight-binding inhibitors confirmed that the formation of the FXIa:PN2KPI complex occurs in a single step that is slow, whereas formation of FXIa:PN2KPI-R15K, FXIa:BPTI and FXIa:BPTI-K15R complexes do not conform to the same kinetic mechanism. Thus PN2KPI and BPTI inhibit FXIa by distinctly different mechanisms and the Arg at the P1 site of PN2KPI confers specificity and high affinity for FXIa inhibition and in part determines the mechanism of PN2KPI as a slow, tight binding inhibitor.

Binding of the bovine basic pancreatic trypsin inhibitor (Kunitz) to human Lys77-plasmin

1986 ◽  
Vol 191 (2) ◽  
pp. 295-297 ◽  
Author(s):  
Enea Menegatti ◽  
Mario Guarneri ◽  
Martino Bolognesi ◽  
Paolo Ascenzi ◽  
Gino Amiconi
Keyword(s):  
Trypsin Inhibitor ◽  
Basic Pancreatic Trypsin Inhibitor ◽  
Pancreatic Trypsin Inhibitor

scholarly journals Free Energy Landscape and Rate Estimation of the Aromatic Ring Flips in Basic Pancreatic Trypsin Inhibitor Using Metadynamics

2021 ◽  
Author(s):  
Pär Söderhjelm ◽  
Mandar Kulkarni
Keyword(s):  
Free Energy ◽  
Trypsin Inhibitor ◽  
Side Chain ◽  
Side Chains ◽  
Aromatic Residues ◽  
Basic Pancreatic Trypsin Inhibitor ◽  
Pancreatic Trypsin Inhibitor ◽  
Energy Surfaces ◽  
Aromatic Side Chains

Aromatic side-chains (phenylalanine and tyrosine) of a protein flip by 180° around the Cβ-Cγ axis (χ2 dihedral of side-chain) producing two symmetry-equivalent states. The ring-flip dynamics act as an NMR probe to understand local conformational fluctuations. Ring-flips are categorized as slow (ms onwards) or fast (ns to near ms) based on timescales accessible to NMR experiments. In this study, we investigated the ability of the infrequent metadynamics approach to discriminate between slow and fast ring-flips for eight individual aromatic side-chains (F4, Y10, Y21, F22, Y23, F33, Y35, F45) of basic pancreatic trypsin inhibitor (BPTI). Well-tempered metadynamics simulations were performed to observe ring-flipping free energy surfaces for all eight aromatic residues. The results indicate that χ2 as a standalone collective variable (CV) is not sufficient to classify fast and slow ring-flips. Most of the residues needed χ1 (N−Cχα) as a complementary CV, indicating the importance of librational motions in ring-flips. Multiple pathways and mechanisms were observed for residues F4, Y10, and F22. Recrossing events are observed for residues F22 and F33, indicating a possible role of friction effects in the ring-flipping. The results demonstrate the successful application of the metadynamics based approach to estimate ring-flip rates of aromatic residues in BPTI and identify certain limitations of the approach.

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