RESUMEN
Factor Xa (FXa) hydrolyzes two peptide bonds in prothrombin having (Glu/Asp)-Gly-Arg-(Thr/Ile) for P(3)-P(2)-P(1)-P(1)' residues, but the exact preferences of its catalytic groove remain largely unknown. To investigate the specificity of FXa, we synthesized full sets of fluorescence-quenched substrates carrying all natural amino acids (except Cys) in P(3), P(2), P(1)', P(2)', and P(3)' and determined the k(cat)/K(m) values of cleavage. Contrary to expectation, glycine was not the "best" P(2) residue; peptide with phenylalanine was cleaved slightly faster. In fact, FXa had surprisingly limited preferences, barely more pronounced than trypsin; in P(2), the ratio of the k(cat)/K(m) values for the most favorable side chain over the least was 289 (12 with trypsin), but in P(1)', this ratio was only 30 (versus 80 with trypsin). This unexpected selectivity undoubtedly distinguished FXa from thrombin, which exhibited ratios higher than 19,000 in P(2) and P(1)'. Thus, with respect to the catalytic groove, FXa resembles a low efficiency trypsin rather than the highly selective thrombin. The rates of cleavage of the peptidyl substrates were virtually identical whether or not FXa was in complex with factor Va, suggesting that the cofactor did not exert a direct allosteric control on the catalytic groove. We conclude that the remarkable efficacy of FXa within prothrombinase originates from exosite interaction(s) with factor Va and/or prothrombin rather than from the selectivity of its catalytic groove.
Asunto(s)
Dominio Catalítico , Factor Xa/metabolismo , Secuencia de Aminoácidos , Factor Xa/química , Humanos , Cinética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Trombina/metabolismo , Tripsina/metabolismoRESUMEN
The control of coagulation enzymes by antithrombin is vital for maintenance of normal hemostasis. Antithrombin requires the co-factor, heparin, to efficiently inhibit target proteinases. A specific pentasaccharide sequence (H5) in high affinity heparin induces a conformational change in antithrombin that is particularly important for factor Xa (fXa) inhibition. Thus, synthetic H5 accelerates the interaction between antithrombin and fXa 100-fold as compared with only 2-fold versus thrombin. We built molecular models and identified residues unique to the active site of fXa that we predicted were important for interacting with the reactive center loop of H5-activated antithrombin. To test our predictions, we generated the mutants E37A, E37Q, E39A, E39Q, Q61A, S173A, and F174A in human fXa and examined the rate of association of these mutants with antithrombin in the presence and absence of H5. fXa(Q61A) interacts with antithrombin alone with a nearly normal k(ass); however, we observe only a 4-fold increase in k(ass) in the presence of H5. The x-ray crystal structure of fXa reveals that Gln(61) forms part of the S1' and S3' pocket, suggesting that the P' region of the reactive center loop of antithrombin is crucial for mediating the acceleration in the rate of inhibition of fXa by H5-activated antithrombin.