Distortion of the catalytic domain of tissue-type plasminogen activator by plasminogen activator inhibitor-1 coincides with the formation of stable serpin-proteinase complexes.
Plasminogen activator inhibitor-1 (PAI-1) is a typical member of the serpin family that kinetically traps its target proteinase as a covalent complex by distortion of the proteinase domain. Incorporation of the fluorescently silent 4-fluorotryptophan analog into PAI-1 permitted us to observe changes in the intrinsic tryptophan fluorescence of two-chain tissue-type plasminogen activator (tPA) and the proteinase domain of tPA during the inhibition reaction. We demonstrated three distinct conformational changes of the proteinase that occur during complex formation and distortion. A conformational change occurred during the initial formation of the non-covalent Michaelis complex followed by a large conformational change associated with the distortion of the proteinase catalytic domain that occurs concurrently with the formation of stable proteinase-inhibitor complexes. Following distortion, a very slow structural change occurs that may be involved in the stabilization or regulation of the trapped complex. Furthermore, by comparing the inhibition rates of two-chain tPA and the proteinase domain of tPA by PAI-1, we demonstrate that the accessory domains of tPA play a prominent role in the initial formation of the non-covalent Michaelis complex.
D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015843:Serpins / Q000737:chemistry*; D013997:Time Factors; D010959:Tissue Plasminogen Activator / Q000737:chemistry*; D014364:Tryptophan / Q000031:analogs & derivatives* / Q000737:chemistry
D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D015843:Serpins; C039390:4-fluorotryptophan; D014364:Tryptophan; D010450:Endopeptidases; D010959:Tissue Plasminogen Activator