title: Viscous drag as the source of active site perturbation during protease translocation: insights into how inhibitory processes are controlled by serpin metastability.
abstract: The native form of serine protease inhibitors (serpins) is kinetically trapped in a metastable state, which is thought to play a central role in the inhibitory mechanism. The initial binding complex between a serpin and a target protease undergoes a conformational change that forces the protease to translocate toward the opposite pole. Although structural determination of the final stable complex revealed a detailed mechanism of keeping the bound protease in an inactive conformation, it has remained unknown how the serpin exquisitely translocates a target protease with an acyl-linkage unhydrolyzed. We previously suggested that the acyl-linkage hydrolysis is strongly suppressed by active site perturbation during the protease translocation. Here, we address what induces the transient perturbation and how the serpin metastability contributes to the perturbation. Inhibitory activity of alpha1-antitrypsin (alpha1AT) toward elastase showed negative correlations with medium viscosity and Stokes radius of elastase moiety, indicating that viscous drag directly affects the protease translocation. Stopped-flow measurements revealed that the change in the inhibitory activity is primarily caused by the change in the translocation rate. The native stability of alpha1AT cavity mutants showed a negative correlation with the translocation rate but a positive correlation with the acyl-linkage hydrolysis rate, suggesting that the two kinetic steps are not independent but closely related. The degree of active site perturbation was probed by amino acid nucleophiles, supporting the view that the changes in the acyl-linkage hydrolysis rate are due to different perturbation states. These results suggest that the active site perturbation is caused by local imbalance between a pulling force driving protease translocation and a counteracting viscous drag force. The structural architecture of serpin metastability seems to be designed to ensure the active site perturbation by providing a sufficient pulling force, so the undesirable hydrolytic activity of protease is strongly suppressed during the translocation.
authors.affiliation: Department of Chemical Engineering, PO Box 43121, Texas Tech University, Lubbock, TX 79424, USA. jongshik.shin@ttu.edu
authors.forename: Jong-Shik
authors.initials: JS
authors.lastname: Shin
authors.forename: Myeong-Hee
authors.initials: MH
authors.lastname: Yu
mesh_terms: D000818:Animals; D001665:Binding Sites; D004795:Enzyme Stability; D005990:Glycerol / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000378:metabolism; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014783:Viscosity; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D015842:Serine Proteinase Inhibitors; D015843:Serpins; D000515:alpha 1-Antitrypsin; D010196:Pancreatic Elastase; D005990:Glycerol