ALDH7A1 Enzymatic Activity Assay

ALDH7A1 enzymatic activity was measured by monitoring NADH production at a wavelength of 340 nm using an Agilent 8453 Hewlett-Packard G1103A spectrophotometer. Each assay was performed at 20°C for 300 seconds with measurements taken at 3-second intervals. The reaction assay buffer was 50 mM pyrophosphate buffer at pH 8.0. Enzyme stock solutions were prepared by diluting to the desired concentration with 50 mM pyrophosphate buffer at pH 8.0 supplemented with 5 mM NAD⁺. AASAL was used as the variable substrate (10 μM – 3000 μM) with NAD⁺ at a fixed saturating concentration (2.5 mM). AASAL was synthesized and quantitated as previously reported [8 (link)]. Buffer, NAD⁺, and AASAL were mixed by inversion in a 1 cm 284 QS quartz cuvette (Fisher Scientific) and blanked before addition of enzyme. The final enzyme concentrations in the assays were as follows: 0.07 μM wild-type, 2.2 μM N167S, 0.36 μM P169S, 3.3 μM A171V, 1.1 μM W175A, and 0.36 μM W175G. Triplicate data sets were collected, and kinetic constants were estimated by globally fitting the initial rate data to the Michaelis-Menten equation using Origin 2017. It was not possible to saturate A171V and P169S with AASAL; in these cases, linear regression was applied, and the slope of the best-fit line was interpreted as an approximation of k_cat/K_m.