Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.
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.
Oliva
M L V
ML
Department of Biochemistry, Universidade Federal de Saõ Paulo, Escola Paulista de Medicina, Rua Três de Mayo 100, 04044-020 Saõ Paulo, SP, Brazil. maysa.bioq@epm.br
Andrade
S A
SA
Juliano
M A
MA
Sallai
R C
RC
Torquato
R J
RJ
Sampaio
M U
MU
Pott
V J
VJ
Sampaio
C A M
CA
D000595:Amino Acid Sequence; D000818:Animals; D036481:Bauhinia / Q000737:chemistry*; D001665:Binding Sites; D002417:Cattle; D015951:Factor Xa / Q000737:chemistry; D065427:Factor Xa Inhibitors; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012639:Seeds / Q000737:chemistry; D017385:Sequence Homology; D015842:Serine Proteinase Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D065427:Factor Xa Inhibitors; D005456:Fluorescent Dyes; D010940:Plant Proteins; D015842:Serine Proteinase Inhibitors; D015951:Factor Xa