MAPPING THE ACTIVE-SITE OF FACTOR XA BY SELECTIVE INHIBITORS - AN NMRAND MD STUDY

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH2 and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH2, in the active site of t he blood clotting enzyme factor Zia was determined by using transferre d nuclear Overhauser effect nuclear magnetic resonance (NMR) spectrosc opy, They represent a family of peptidic inhibitors obtained by the sc reening of a vast combinatorial library. Each structure was first calc ulated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the a ctive site and repeating the simulated annealing and energy minimizati on. The final structure was optimized by molecular dynamics simulation s of the inhibitor complex in water, The NMR restraints were kept thro ughout the refinement, The inhibitors assume a compact, very well defi ned conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the pepti des and to map the active site. (C) 1998 Wiley-Liss, Inc.