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An SPR-based method for Hill coefficient measurements: the case of insulin-degrading enzyme

Abstract

Insulin-degrading enzyme (IDE) is a highly conserved zinc metallopeptidase and is capable to catalytically cleave several substrates besides insulin, playing a pivotal role in several different biochemical pathways. Although its mechanism of action has been widely investigated, many conundrums still remain, hindering the possibility to rationally design specific modulators which could have important therapeutical applications in several diseases such as diabetes and Alzheimer’s disease. In this scenario, we have developed a novel surface plasmon resonance (SPR) method which allows for directly measuring the enzyme cooperativity for the binding of insulin in the presence of different IDE activity modulators: carnosine, ATP, and EDTA. Results indicate that both positive and negative modulations of the IDE activity can be correlated to an increase and a decrease of the measured Hill coefficient, respectively, giving a new insight into the IDE activity mechanism. The use of the IDE R767A mutant for which oligomerization is hindered confirmed that the positive allosteric modulation of IDE by carnosine is due to a change in the enzyme oligomeric state occurring also for the enzyme immobilized on the gold SPR chip.

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Acknowledgements

Prof. Massimo Coletta and Prof. Andrea Bellelli are thanked for fruitful discussion and advices.

Funding

This research was supported by University of Catania Programma Ricerca di Ateneo Unict 2020–2022-Linea 2 (Project: 3 N-ORACLE). Alessia Distefano and Gabriele Antonio Zingale were supported by the PhD program in Chemical Sciences, University of Catania.

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Correspondence to Giuseppe Grasso.

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Distefano, A., Antonio Zingale, G. & Grasso, G. An SPR-based method for Hill coefficient measurements: the case of insulin-degrading enzyme. Anal Bioanal Chem 414, 4793–4802 (2022). https://doi.org/10.1007/s00216-022-04122-3

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  • DOI: https://doi.org/10.1007/s00216-022-04122-3

Keywords

  • Enzymes
  • Kinetics
  • Biosensors
  • Bioassays
  • Bioanalytical methods