Kinetic mechanism of protease inhibition by alpha1-antitrypsin.
The native form of serine protease inhibitor (serpin) is kinetically trapped in a metastable state. Metastability in these proteins is critical to inhibit target protease by forming a stable covalent complex. Despite recent determination of the crystal structures of a Michaelis protease-serpin complex as well as a stable covalent complex, details on the kinetic mechanism remain unsolved. In this report, we examined the reaction mechanism of alpha1-antitrypsin toward elastase by a combination of stopped-flow experiments via fluorescence resonance energy transfer and rapid-quench studies. The results suggest a non-covalent complex intermediate other than Michaelis complex as an intermediate before the cleavage of P1-P1' scissile bond, whose formation is the rate-determining step of the overall reaction. This rate-limiting step represents rearrangement of the reactive site loop, and is regulated by a salt bridge between E354 and R196. The ionic interaction is unique to alpha1-antitrypsin, which suggests that protease inhibition mechanisms are varied among serpins.
Kang
Un-Beom
UB
Biomedical Research Center, Korea Institute of Science and Technology, P.O. Box 131, Cheongryang, Seoul 130-650, Republic of Korea.
Baek
Je-Hyun
JH
Ryu
Seung-Hyun
SH
Kim
Joon
J
Yu
Myeong-Hee
MH
Lee
Cheolju
C
D019943:Amino Acid Substitution; D001665:Binding Sites; D004789:Enzyme Activation; D004791:Enzyme Inhibitors; D017022:Flow Injection Analysis; D007700:Kinetics; D010196:Pancreatic Elastase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D000515:alpha 1-Antitrypsin / Q000737:chemistry*
D004791:Enzyme Inhibitors; D000515:alpha 1-Antitrypsin; D010196:Pancreatic Elastase