Modulation of serpin reaction through stabilization of transient intermediate by ligands bound to alpha-helix F.
Mechanism-based inhibition of proteinases by serpins involves enzyme acylation and fast insertion of the reactive center loop (RCL) into the central beta-sheet of the serpin, resulting in mechanical inactivation of the proteinase. We examined the effects of ligands specific to alpha-helix F (alphaHF) of plasminogen activator inhibitor-1 (PAI-1) on the stoichiometry of inhibition (SI) and limiting rate constant (k(lim)) of RCL insertion for reactions with beta-trypsin, tissue-type plasminogen activator (tPA), and urokinase. The somatomedin B domain of vitronectin (SMBD) did not affect SI for any proteinase or k(lim) for tPA but decreased the k(lim) for beta-trypsin. In contrast to SMBD, monoclonal antibodies MA-55F4C12 and MA-33H1F7, the epitopes of which are located at the opposite side of alphaHF, decreased k(lim) and increased SI for every enzyme. These effects were enhanced in the presence of SMBD. RCL insertion for beta-trypsin and tPA is limited by different subsequent steps of PAI-1 mechanism as follows: enzyme acylation and formation of a loop-displaced acyl complex (LDA), respectively. Stabilization of LDA through the disruption of the exosite interactions between PAI-1 and tPA induced an increase in the k(lim) but did not affect the SI. Thus it is unlikely that LDA contributes significantly to the outcome of the serpin reaction. These results demonstrate that the rate of RCL insertion is not necessarily correlated with SI and indicate that an intermediate, different from LDA, which forms during the late steps of PAI-1 mechanism, and could be stabilized by ligands specific to alphaHF, controls bifurcation between the inhibitory and the substrate pathways.
Komissarov
Andrey A
AA
Department of Chemistry, Portland State University, Portland, Oregon 97207-0751, USA. akomiss@pdx.edu
Zhou
Aiwu
A
Declerck
Paul J
PJ
D000215:Acylation; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008024:Ligands; D008956:Models, Chemical; D008958:Models, Molecular; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry*; D019096:Vitronectin / Q000737:chemistry / Q000235:genetics
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008024:Ligands; D017395:Plasminogen Activator Inhibitor 1; D015842:Serine Proteinase Inhibitors; D019096:Vitronectin; D014357:Trypsin; D010959:Tissue Plasminogen Activator