"switching On" Enzyme Substrate Specificity Analysis with a Fluorescent Competitive Inhibitor

Alex Strom, Rachit Shah, Carston R. Wagner

Research output: Contribution to journalArticlepeer-review


Enzymatically driven change to the spectroscopic properties of a chemical substrate or product has been a linchpin in the development of continuous enzyme kinetics assays. These assays inherently necessitate substrates or products that naturally comply with the constraints of the spectroscopic technique being used, or they require structural changes to the molecules involved to make them observable. Here we demonstrate a new analytical kinetics approach with enzyme histidine triad nucleotide binding protein 1 (HINT1) that allows us to extract both useful kcat values and a rank-ordered list of substrate specificities without the need to track substrates or products directly. Instead, this is accomplished indirectly using a "switch on"competitive inhibitor that fluoresces maximally only when bound to the HINT1 enzyme active site. Kinetic information is extracted from the duration of the diminished fluorescence when the monitorable inhibitor-bound enzyme is challenged with saturating concentrations of a nonfluorescent substrate. We refer to the loss of fluorescence, while the substrate competes for the fluorescent probe in the active site, as the substrate's residence transit time (RTT). The ability to assess kcat values and substrate specificity by monitoring the RTTs for a set of substrates with a competitive "switch on"inhibitor should be broadly applicable to other enzymatic reactions in which the "switch on"inhibitor has sufficient binding affinity over the enzymatic product.

Original languageEnglish (US)
Pages (from-to)440-450
Number of pages11
Issue number6
StatePublished - Feb 16 2021

Bibliographical note

Funding Information:
This work was funded by the University of Minnesota Foundation with additional support from the American Foundation for Pharmaceutical Education predoctoral fellowship awarded to A.S.

Publisher Copyright:
© 2021 American Chemical Society.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't


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