Studies of enzymes from two protease families: Tissue Kallikreins, ADAMs and MMPs.

摘要: The human kallikrein family is a of proteolytic enzymes, classified as serine proteases, that derive from chromosome 19, locus 13.3-13.4. These enzymes are widespread in pathophysiological processes such cancer and neurodegenerative diseases; hence studies catalytic sites inhibitors important relation to the longer term design therapeutic drugs. One member family, 4 (hK4) which thought carry out crucial functions prostate, was expressed this study secreted protein baculovirus expression system, bearing His-tag V5-epitope were used for purification detection respectively. Its mass estimated be 35kDa, ~2kDa less than equivalent product monkey kidney cells. purified 50-90% purity with yield 0.93mg/L-4.8mg/L based on methods derived computational prediction its properties, pI. Computational analysis extended by applying high-performing computing techniques, molecular dynamics, flexible ligand docking, predict antigenic regions, likely substrate specificity putative inhibitors. results show hK4 has loop, between Leu83-Ser94 shows promise specific segment can exploited generation antibodies. Preferred substrates also predicted bear hydrophobic residues at P'-region scissile bond amphiphilic P-region. At S-region, potentially involves unique PLYH-motif recognizing P4/P5 position substrate. Flexible ligand-docking indicate inhibited modified bulky sidechain guanidinium group P1-position own autoactivation region P2, P1' P2' position. other members predicting distinctions these could future studies. 8 fifteen very homologous terms typical trypsinlike activity specificity, except hK2, hK3, hK4, hK5, hK7, hK9, hK15 retain certain distinct signatures binding pocket secondary specificity. principles substrate-specificity developed further applied three metzincins, MMP-3, ADAM-9 ADAM-10. metalloproteases, involved tissue remodeling, intracellular signalling cell-to-cell mediation. carried all metzincins using structure crystallized complex MMP-3 enzyme TIMP-1 natural inhibitor template. In enzyme-substrate complex, challenge model suggest possible orientation P-region, not known. interactions P/S-region therefore unclear need clarified. order arrangement undefined S-subsites, four new added an beta-sheet conformation residue (derived originally inhibitor) create full-length modeled spanning P4'-P4. This region, particular, bound through backbone H-bonds 169 (MMP nomenclature) suggesting binding, satisfied steric chemical restraints S'-region enzyme. modeling approach indicated presence S2/S3-pocket composed different ADAM-10, prove useful drug projects. Furthermore, data argue against involvement polarizable water molecule catalysis, mechanism been postulated various groups. A suggested involve oxyanion anhydride transition state. demonstration power combining bioinformatics wet-lab biochemistry.