Enzyme activity of Aquincola tertiaricarbonis L108 acylating aldehyde dehydrogenase ATN38601 against various aldehyde and acyl-CoA substrates

Method
Kinetic assays were performed using a photometer (Hitachi U-2000 Spectrophotometer, in SUPRASIL quartz cuvettes (path length 10 mm) at 35°C (or temperature as indicated). As assay buffer a solution of 50 mM HK2PO4/H2KPO4, 50 mM Tris/HCl, 1 mM DTT, 20% glycerol, pH 7.8 (or at pH as indicated) was used. For quantifying the oxidation (Ox) of aldehydes, each 400 µL reaction contained buffer, 2 mM NAD+ (or as indicated), 5 mM CoA (or as indicated), purified enzyme (heterologously expressed in E. coli, N-terminal 6xHis tag; wild type = WT, enzyme variants with A254I, A254P or A254G replacements) at concentrations as indicated and various concentrations of aldehydes. For quantifying the reduction (Red) of acyl-CoAs, each 400 µL reaction contained buffer, 0.625 mM NADH (or as indicated), purified enzyme (WT) at concentrations as indicated and various concentrations of acyl-CoAs. The enzyme activity was monitored by measuring the formation/consumption of NADH at 340 nm using an absorption coefficient of 6.22 mM-1 cm-1. Enzyme activity (U) is expressed as µmol NADH produced/consumed per minute. Steady state kinetic data, obtained with technical repeats, were analyzed by non-linear regression to the Michaelis-Menten (MM) equation using GraphPad Prism software.

Data
The following data sets are included in this collection:

Temperature and pH optima
Co-factor Km values
Kinetic Data for aldehydes and acyl-CoAs

Raw data processing method

The following method was used to process the data:

-Export raw data from photometer (see file Assay_Rawdata.txt and corresponding meta data file Assay_Metadata.txt)
-Analyze slope (Abs340nm/s) by linear regression
-Calculate enzyme activity in U/mg enzyme
-Calculate mean and SD for technical replicates
-Analyze by nonlinear regression (curve fit, MM).