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. 2020 Aug 12;10:13621. doi: 10.1038/s41598-020-70598-7

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Characterization of PLP inhibition. (a) The initial velocity of the reaction was measured with 2 µM enzyme (protein subunit concentration), varying PNP concentration while keeping exogenous PLP fixed at different concentrations (0, 0.25, 0.5, 1, 2, 4, 8 and 16 µM, as indicated by the pointing down arrow). Data are the average ± standard deviation of three independent measurements. The Michaelis–Menten equation was fitted to the resulting saturation curves, obtaining apparent V_max and K_M values at all PLP concentrations. (b) Fitting of 1/V_max (blue symbols) and K_M/V_max (red symbols), obtained from the fitting of data shown in panel A, using an increasing hyperbolic equation and a parabolic equation, respectively. (c) Initial velocity data rearranged as a function of PLP concentration, and obtained at different, fixed substrate concentrations (v_X). The increasing PNP concentration is indicated by the pointing up arrow. Continuous lines result from global fitting of v_x values to Eq. (3), as detailed in the Methods section, which gave the parameters reported in the text. Images were generated using the software Prism 8 (GraphPad; https://www.graphpad.com/scientific-software/prism/).