Factor Xa Active Site Substrate Specificity with Substrate Phage Display and Computational Molecular Modeling*

Structural origin of substrate-enzyme recognition remains incompletely understood. In themodel enzyme system of serine protease, canonical anti-parallel -structure substrate-enzyme complex is the predominant hypothesis for the substrate-en-zyme interaction at the atomic level. We used factor Xa (fXa), a key serine protease of the coagulation system, as a model enzyme to test the canonical conformation hypothesis. More than 160 fXa-cleavable substrate phage variants were experi-mentally selected from three designed substrate phage display libraries. These substrate phage variants were sequenced and their specificities to the model enzyme were quantified with quantitative enzyme-linked immunosorbent assay for substrate phage-enzyme reaction kinetics. At least three substrate-en-zyme recognitionmodes emerged from the experimental data as necessary to account for the sequence-dependent specificity o