Kinetic Characterization of Factor Xa Binding Using a Quenched Fluorescent Substrate Based on the Reactive Site of Factor Xa Inhibitor from Bauhinia ungulata Seeds

Abstract

The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

Structural and inhibitory properties of a plant proteinase inhibitor containing the RGD motif

“…The alignment of BrTI amino acid sequence with those of other Bauhinia inhibitors shows their high degree of conservation throughout the evolution of this genus [16][17][18][19][20][21]. In particular, many conserved regions in plant-Kunitz type inhibitors are present despite of the absence of disulfide bridges characteristic in other Bauhinia species, such as Arg 64 at P1 position of the reactive site, Asp 5 , Gly 8 , Pro 10 , Asn 13 , Tyr-Tyr 17,18 , Gly 28 , Gly-Asn-Glu 35-37 (SbTI numbering) [29].…”

“…The peptide YLEPVARGDGGLA-NH 2 comprising part the original BrTI sequence (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31), and two other peptides with Asp replaced by Glu, and N-terminal Glu replaced by Ile, respectively YLEPVARGEGGLA-NH 2 and YLIPVARGDGGLA-NH 2 , were synthesized and comparatively studied. The stability and purification of the peptides were periodically assessed by its retention time on a reverse phase column.…”

“…The inhibitors appear as a single polypeptide chain of molecular mass 20 kDa and they show structural similarity to other plant Kunitz inhibitor family [17][18][19]. We characterized inhibitors with two disulfide bridges from B. varigata (BvTI) and from B. ungulata (BuXI), similar to the classical plant Kunitz, STI [20]. An intermediate form represented by gBrTI isolated from Bauhinia rufa has one disulfide bridge and a glycosilated site [21].…”

Bauhinia bauhinioides cruzipain inhibitor reduces endothelial proliferation and induces an increase of the intracellular Ca2+ concentration

“…in plants defence mechanism against pests and pathogens [24,37]. The best characterized serine PI families are the Bowman-Birk inhibitors and the Kunitz-type inhibitors, which are isolated from many leguminous plants like the genus Bauhinia [7,32,33]. Proteinase inhibitors from Bauhinia bauhinioides are known to effect physiological processes like blood coagulation, inflammation or cancer [29,30,38].…”

Interaction of proteinase inhibitors with phospholipid vesicles is modulated by pH

“…Due to their actions on target enzymes in a diverse array of biological processes, these proteins are useful tools for the investigation of digestion [2,3], coagulation [4,5], inflammation [6,7], cancer [8][9][10] and fibrinolysis [11,12]. All of these events involve a complex meshwork of activities.…”