The thioredoxin system and not the Michaelis–Menten equation should be fitted to substrate saturation datasets from the thioredoxin insulin assay

Abstract

Introduction: The thioredoxin system, consisting of thioredoxin reductase, thioredoxin and NADPH, is present in most living organisms and reduces a large array of target protein disulfides.

Objective: The insulin reduction assay is commonly used to characterise thioredoxin activity in vitro, but it is not clear whether substrate saturation datasets from this assay should be fitted and modeled with the Michaelis-Menten equation (thioredoxin enzyme model), or fitted to the thioredoxin system with insulin reduction described by mass-action kinetics (redox couple model).

Methods: We utilized computational modeling and in vitro assays to determine which of these approaches yield consistent and accurate kinetic parameter sets for insulin reduction.

Results: Using computational modeling, we found that fitting to the redox couple model, rather than to the thioredoxin enzyme model, resulted in consistent parameter sets over a range of thioredoxin reductase concentrations. Furthermore, we established that substrate saturation in this assay was due to the progressive redistribution of the thioredoxin moiety into its oxidised form. We then confirmed these results in vitro using the yeast thioredoxin system.

Discussion: This study shows how consistent parameter sets for thioredoxin activity can be obtained regardless of the thioredoxin reductase concentration used in the insulin reduction assay, and validates computational systems biology modeling studies that have described the thioredoxin system with the redox couple modeling approach.

Keywords:

• Redoxin
• Systems biology
• Redox regulation
• Kinetic model

Acknowledgments

The authors wish to thank Professor Johann M. Rohwer (Stellenbosch University) for generously providing the modeling scripts used in this study and for critical comments on the revised manuscript.

Disclaimer statements

Contributors LP and CSP performed the modeling analysis. LP performed the experimental work and drafted the paper while CSP edited the paper.

Funding This work is based on the research supported in part by the National Research Foundation (NRF) of South Africa. Any opinion, finding and conclusion or recommendation expressed in this material is that of the author(s) and the NRF does not accept any liability in this regard. LP is a recipient of an NRF Scarce Skills Scholarship.

Conflicts of interest The authors declare no conflict of interest.

Ethics approval Ethical approval was not required for this study.

Supplementary material

Supplementary material for this article can be found at: www.maneyonline.com/doi/suppl/10.1179/1351000215Y.0000000024.