Abstract
Protease inhibitors are crucial for the control of proteolytic activity in different physiological processes. However, some inhibitors do not show canonical enzyme recognition of the enzyme under certain conditions. In this work, we present evidence that indicates the formation of an active complex between the protease bovine α-chymotrypsin and the Tepary bean protease inhibitor (TBPI). The composition of the active chymotrypsin-TBPI complex (AC) was confirmed by three different methods: size-exclusion chromatography, polyacrylamide gel electrophoresis (PAGE), and mass spectrometry. The kinetic parameters for the AC were similar to those of the enzyme alone, indicating that TBPI binding does not produce any large changes in chymotrypsin. The molecular model proposed here postulates that TBPI binds outside the active cleft of the protease, but near enough to hinder the binding of high molecular weight substrates into the active site. This model was experimentally supported by the inhibitory effect on casein as a substrate, and the unaltered protease activity when a small synthetic substrate was used. We also found that the formation of this complex provided the enzyme with extra stability in denaturing conditions or in the presence of a reducing agent. The chymotrypsin-TBPI complex exhibited higher stability, indicating that autolysis can be partially prevented. When the enzyme was first inactivated followed by the addition of the inhibitor, the activity of the protease was restored. We described a possible mechanism where a plant protease inhibitor binds outside the active site of the enzyme while increasing its stability.
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Abbreviations
- AC:
-
Active chymotrypsin-TBPI complex
- appMM:
-
Apparent molecular mass
- AU:
-
Proteolytic activity units
- BApNA:
-
N-benzoyl-arginine-p-nitroanilide
- BBI:
-
Bowman-Birk type inhibitor
- BSA:
-
Bovine serum albumin
- Da:
-
Daltons
- DMSO:
-
Dimethyl sulfoxide
- DTT:
-
Dithiothreitol
- E:
-
Enzyme/α-chymotrypsin
- EI:
-
Enzyme-Inhibitor/chymotrypsin-TBPI
- IC:
-
Inactive chymotrypsin-TBPI complex
- IU:
-
Inhibition activity units
- Km:
-
Michaelis–Menten constant
- M:
-
Molar
- MALDI-TOF-TOF:
-
Matrix assisted laser desorption ionization time-of-flight
- MMP:
-
Matrix metalloproteinase
- MS/MS:
-
Tandem mass spectrometry
- PAGE:
-
Polyacrylamide gel electrophoresis
- PI:
-
Protease inhibitor
- PI’s:
-
Protease inhibitors
- SAAFpNA:
-
N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide
- SDS:
-
Sodium dodecyl sulfate
- SDS-PAGE:
-
Polyacrylamide gel electrophoresis with sodium dodecyl sulfate
- TBPI:
-
Tepary bean protease inhibitor
- TEMED:
-
1,2-bis(dimethylamine)-ethane
- Tm:
-
Melting temperature
- Vmax:
-
Maximum velocity
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Acknowledgements
We thank CONACYT grant 182760ABL, and fellowships of R Pliego-Arreaga, O Roldán-Padrón and M Dagio-Hernández. To Alicia Chagolla for her efficient assistance with mass spectrometry. Thanks to Dr. Jorge A. Torres-Castillo, and also thanks to Dr. Collen Beard and Carolyn Smith of Peace Corps Response for their useful advice when reviewing the manuscript.
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Pliego-Arreaga, R., Roldán-Padrón, O., Castro-Guillén, J.L. et al. Properties of a Non-canonical Complex Formed Between a Tepary Bean (Phaseolus acutifolius) Protease Inhibitor and α-Chymotrypsin. Protein J 38, 435–446 (2019). https://doi.org/10.1007/s10930-019-09863-2
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DOI: https://doi.org/10.1007/s10930-019-09863-2