Abstract
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.
Highlights
Serpins are a family of mainly serine proteinase inhibitors, some members inhibit cysteine proteinases whereas others have evolved into non-inhibitory forms such as hormone carriers and immunomodulatory factors (1)
Using stopped flow kinetics and fluorescence resonance energy transfer (FRET) between fluorescein-labeled elastase and tetramethylrhodamine-labeled ␣1-antitrypsin, we came to the conclusion that the minimum scheme describing this serpin-proteinase interaction was as described in the following scheme (13)
We further studied the reversible steps characterized by successive conformational changes
Summary
Serpins are a family of mainly serine proteinase inhibitors, some members inhibit cysteine proteinases whereas others have evolved into non-inhibitory forms such as hormone carriers and immunomodulatory factors (1). The position of the proteinase on the top of the uncleaved and uninserted loop suggests that it provides a good model of the first reversible encounter complex, the so-called Michaelis-type complex, which has previously been studied kinetically (9). This complex is converted to the final complex via multiple steps, including irreversible acylation and several reversible steps (10 –12). The conformational changes that diagnose the occurrence of reversible intermediate steps are of great importance regarding the comprehension of how serpins regulate subtle proteolytic cascades They determine the interval of time during which the inhibition is reversible. FRET measurements at equilibrium were correlated with time-resolved fluorescence measurements for a better precision and to be able to analyze mixtures of species
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
