The dataset refers to a single manuscript in which three different fluorescent substrates were used to assess the enzymatic activity of purified ALDH isoforms and their application to detect ALDH in low-activity cell extracts. Raw data corresponding to Tables 1, 2, and 3, Figures 5 and 6, and supplementary data Figure S3.

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Instruments, calibration and standards information - Cary Eclipse Fluorescence spectrometer (Agilent). 5 ?M NADH/ NADPH was used as an internal Standard. The absorbance spectrum of BAAA was obtained using a Cary 400 Varian UV–Visible spectrophotometer. The molar extinction coefficient was calculated from the linear plot of absorbance versus known concentrations of the compound, resulting in ?506 =33.2 ×103 M-1·cm-1. This allowed us to determine the precise substrate concentration prior to each experiment from the absorbance measurement in commercial Assay Buffer. A Cary 400 Varian UV–vis spectrophotometer was used to determine the absorbance spectra of MONAL-62, 6-methoxy-2-naphthoic acid (MONOIC-62), MONAL-71, 7-methoxy-1-naphthoic acid (MONOIC-71) and NADH in water:acetonitrile (60:40, v/v) for MONAL series of compounds and 50 mM HEPES, pH 8.0, for NADH. Precise substrate concentration was assessed from molar extinction coefficients for MONAL-62, ?316 =14.2 ×103 M-1·cm-1, and MONAL-71, ?352 =6.1 ×103 M-1·cm-1. Environmental or experimental conditions - 25ºC for BAAA and 37ºC for MONAL compounds. Quality-assurance procedures performed on the data - Data are presented as the mean ± standard deviation from duplicate measurements and the kinetic parameters are shown as the mean ± standard error.