AT-P2-Phe inhibited fXa with ～5-fold slower pseudo-first order rate constant (kobs), however, AT-QFRSLS exhibited ～1.5-fold improved reactivity with the protease ( Fig. 2A). The latter results support the amidolytic activity data that
pirinixic acid QFRSLS may be the most preferred P3–P3′ residues for fXa [22]. Nevertheless, the data with AT-P2-Phe further suggests that there are cooperative effects between P3–P3′ residues and that a P2 preference for Phe by fXa is dependent on the nature of other residues surrounding the scissile bond. This mechanism of specificity may explain how fXa can activate other natural substrates (i.e., factors V and VIII) containing bulky residues like Leu and Ile at the P2 positions. Based on structural and modeling data,
phagocytosis has been hypothesized that the phenolyic side chain of Tyr-99 has conformational plasticity to swing out of its position in order to accommodate P2 residues with bulkier side chains at the S2 sub-site [21]. The likely candidate, influencing the P2 recognition specificity of fXa, is the P3 residue since no significant structural differences appear to exist between other P3–P3′ residues of AT-P2-Phe (AFRSLN) and AT-QFRSLS.
