Submitted to: Methods in Enzymology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/2/2022
Publication Date: 11/29/2022
Citation: Sullivan, M.L. 2022. Near-real time determination of BAHD acyl-coenzyme A transferase reaction rates and kinetic parameters using Ellman's reagent. In: Jez, Joseph, editor. Methods in Enzymology. Volume 683. Cambridge, MA: Academic Press. p.19-39.
Interpretive Summary: BAHD acyl-coenzyme A (CoA) acyltransferases are a large class of enzymes involved in many important biochemical processes in plants, including the formation of bioactive compounds with nutraceutical potential. An important way of characterizing what enzymes do is to measure how efficiently they can convert various substrates to products by determining kinetic parameters. To make such determinations, how quickly the enzymes carry out chemical reactions needs to be measured. Making these measurements can be challenging and time consuming. Here we describe a simple procedure for measuring BAHD acyl-CoA acyltransferase reaction rates by detecting the formation of the free CoA reaction product by using Ellman’s reagent, a chemical that changes color when it reacts with the free CoA. This approach will allow researchers studying this class of biosynthetic enzyme to more easily make rate and kinetic measurements in order to understand their functions.
Technical Abstract: BAHD acyl-coenzyme A (CoA) acyltransferases play key roles in a large number of biosynthetic reactions involved in plant specialized metabolism. One approach to measure reaction rates for these enzymes is to quantify the amide or ester reaction products following chromatographic separation of reaction components, an approach that can be labor intensive and time consuming, and complicated by a lack of pure standards. We previously developed and validated an alternative approach using 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB, Ellman’s reagent) to spectrophotometrically monitor reaction progress by the release of free CoA in the reaction. This approach allows near real time measurement of reaction rates, permitting reaction conditions (buffer, reactant and enzyme concentrations, etc.) to be changed “on the fly”. The ease and rapidity of data collection allows a high density of data points to be collected for determination of kinetic parameters. Here we provide a detailed procedure for using DTNB to measure BAHD acyl-CoA acyltransferase reaction rates, and as an example, use it to determine kinetic parameters for red clover hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase, a BAHD acyl-CoA hydroxycinnamoyl transferase not previously characterized with respect to kinetic parameters.