Comparative trajectories of active and S195A inactive trypsin upon binding to serpins.
Serpins inhibit proteinases through a complicated multistep mechanism. The precise nature of these steps and the order by which they occur are still debated. We compared the fate of active and S195A inactive rat trypsin upon binding to alpha(1)-antitrypsin and P(1)-Arg-antichymotrypsin using stopped-flow kinetics with fluorescence resonance energy transfer detection and time-resolved fluorescence resonance energy transfer. We show that inhibition of active trypsin by these serpins leads to two irreversible complexes, one being compatible with the full insertion of the serpin-reactive site loop but not the other one. Binding of inactive trypsin to serpins triggers a large multistep reversible rearrangement leading to the migration of the proteinase to an intermediate position. Binding of inactive trypsin, unlike that of active trypsin, does not perturb the rhodamine fluorescence at position 150 on the helix F of the serpin. Thus, inactive proteinases do not migrate past helix F and do not trigger full serpin loop insertion.
Mellet
Philippe
P
Laboratoire d'Enzymologie, INSERM Unite 392, Universite Louis Pasteur de Strasbourg, F-67400 Illkirch, France.
Mély
Yves
Y
Hedstrom
Lizbeth
L
Cahoon
Marguerite
M
Belorgey
Didier
D
Srividya
Narayanan
N
Rubin
Harvey
H
Bieth
Joseph G
JG
D000818:Animals; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D012235:Rhodamines / Q000378:metabolism; D015843:Serpins / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors; D014357:Trypsin / Q000235:genetics / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism*; D000515:alpha 1-Antitrypsin / Q000378:metabolism*
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D012235:Rhodamines; D015843:Serpins; D014361:Trypsin Inhibitors; D000515:alpha 1-Antitrypsin; C005358:tetramethylrhodamine; D014357:Trypsin