Partitioning of serpin-proteinase reactions between stable inhibition and substrate cleavage is regulated by the rate of serpin reactive center loop insertion into beta-sheet A.
The serpin family of serine proteinase inhibitors is a mechanistically unique class of naturally occurring proteinase inhibitors that trap target enzymes as stable covalent acyl-enzyme complexes. This mechanism appears to require both cleavage of the serpin reactive center loop (RCL) by the proteinase and a significant conformational change in the serpin structure involving rapid insertion of the RCL into the center of an existing beta-sheet, serpin beta-sheet A. The present study demonstrates that partitioning between inhibitor and substrate modes of reaction can be altered by varying either the rates of RCL insertion or deacylation using a library of serpin RCL mutants substituted in the critical P(14) hinge residue and three different proteinases. We further correlate the changes in partitioning with the actual rates of RCL insertion for several of the variants upon reaction with the different proteinases as determined by fluorescence spectroscopy of specific RCL-labeled inhibitor mutants. These data demonstrate that the serpin mechanism follows a branched pathway, and that the formation of a stable inhibited complex is dependent upon both the rate of the RCL conformational change and the rate of enzyme deacylation.
Lawrence
D A
DA
American Red Cross Holland Laboratory, Rockville, Maryland 20855, USA. lawrenced@usa.redcross.org
Olson
S T
ST
Muhammad
S
S
Day
D E
DE
Kvassman
J O
JO
Ginsburg
D
D
Shore
J D
JD
D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D015843:Serpins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D013696:Temperature; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors
D000596:Amino Acids; D017395:Plasminogen Activator Inhibitor 1; D015843:Serpins; D010450:Endopeptidases; D013917:Thrombin