Using singular perturbation theory to determine kinetic parameters in a non-standard coupled enzyme assay

Dalwadi, Mohit P.; Orol, Diego; Walter, Frederik; Minton, Nigel P.; King, John R.; Kovács, Katalin

Abstract

We investigate how to characterize the kinetic parameters of an aminotransaminase using a non-standard coupled (or auxiliary) enzyme assay, where the peculiarity arises for two reasons. First, one of the products of the auxiliary enzyme is a substrate for the primary enzyme and, second, we explicitly account for the reversibility of the auxiliary enzyme reaction. Using singular perturbation theory, we characterize the two distinguished asymptotic limits in terms of the strength of the reverse reaction, which allows us to determine how to deduce the kinetic parameters of the primary enzyme for a characterized auxiliary enzyme. This establishes a parameter-estimation algorithm that is applicable more generally to similar reaction networks. We demonstrate the applicability of our theory by performing enzyme assays to characterize a novel putative aminotransaminase enzyme, CnAptA (UniProtKB Q0KEZ8) from Cupriavidus necator H16, for two different omega-amino acid substrates.

Citation

Dalwadi, M. P., Orol, D., Walter, F., Minton, N. P., King, J. R., & Kovács, K. (2020). Using singular perturbation theory to determine kinetic parameters in a non-standard coupled enzyme assay. Journal of Mathematical Biology, 81(2), 649-690. https://doi.org/10.1007/s00285-020-01524-8

• Journal Article Type: Article
• Acceptance Date: Jul 14, 2020
• Online Publication Date: Aug 6, 2020
• Publication Date: Aug 6, 2020
• Deposit Date: Aug 10, 2020
• Publicly Available Date: Aug 18, 2020
• Journal: Journal of Mathematical Biology
• Print ISSN: 0303-6812
• Electronic ISSN: 1432-1416
• Publisher: Springer Verlag
• Peer Reviewed: Peer Reviewed
• Volume: 81
• Issue: 2
• Pages: 649-690
• DOI: https://doi.org/10.1007/s00285-020-01524-8
• Keywords: Agricultural and Biological Sciences (miscellaneous); Modelling and Simulation; Applied Mathematics
• Public URL: https://nottingham-repository.worktribe.com/output/4816350
• Publisher URL: https://link.springer.com/article/10.1007%2Fs00285-020-01524-8
• Additional Information: Received: 7 August 2019; Revised: 29 June 2020; First Online: 6 August 2020