Heterogeneity in serpin-protease complexes as demonstrated by differences in the mechanism of complex breakdown.
Serpins trap their target proteases in the form of an acyl-enzyme complex. The trap is kinetic, however, and thus serpin-protease complexes ultimately break down, releasing a cleaved inactive serpin and an active protease. The rates of this deacylation process vary greatly depending on the serpin-protease pair with half-lives ranging from minutes to months. The reasons for the diversity in breakdown rates are not clearly understood. In the current study, pH and solvent isotope effects were utilized to probe the mechanism of breakdown for an extremely stable complex and several unstable complexes. Two different patterns for the pH dependence of k(bkdn), the first-order rate constant of breakdown, were found. The stable complex, which breaks down at neutral pH with a half-life of approximately 2 weeks, exhibited a pH-k(bkdn) profile consistent with solvent-hydroxide ion mediated ester hydrolysis. There was no evidence for the participation of the catalytic machinery in the breakdown of this complex, suggesting extensive distortion of the active site. The unstable complexes, which break down with half-lives ranging from minutes to hours, exhibited a bell-shaped pH profile for k(bkdn), typical of the pH-rate profiles of free serine proteases. In the low to neutral pH range k(bkdn) increased with increasing pH in a manner characteristic of His57-mediated catalysis. In the alkaline pH range a decrease in k(bkdn) was observed, consistent with the titration of the Ile16-Asp194 salt bridge (chymotrypsinogen numbering). The alkaline pH dependence was not exhibited in pH-rate profiles of free or substrate-bound HNE, indicating that the salt bridge was significantly destabilized in the complexed protease. These results indicate that breakdown is catalytically mediated in the unstable complexes although, most likely, the protease is not in its native conformation and the catalytic machinery functions inefficiently. However, a mechanism in which breakdown is determined by the equilibrium between distorted and undistorted forms of the complexed protease cannot be completely dismissed. Overall, the results of this study suggest that the protease structure in unstable complexes is distorted to a lesser extent than in stable complexes.
Plotnick
Michael I
MI
Pulmonary, Allergy, and Critical Care Division, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA. mplotnic@mail.med.upenn.edu
Samakur
Madhurika
M
Wang
Zhi Mei
ZM
Liu
Xhuzuo
X
Rubin
Harvey
H
Schechter
Norman M
NM
Selwood
Trevor
T
D002384:Catalysis; D017666:Deuterium Oxide; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009504:Neutrophils / Q000201:enzymology; D010196:Pancreatic Elastase / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D015843:Serpins / Q000737:chemistry / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism; D000514:alpha 1-Antichymotrypsin / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D015843:Serpins; D000514:alpha 1-Antichymotrypsin; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase; D014357:Trypsin; D017666:Deuterium Oxide