hMDH1 Oxaloacetate Kinetic Data
Description
Shown are fits to kinetic data of human cytoplasmic malate dehydrogenase (hMDH1), UniProt ID P40925, with constant concentration of NADH and varying concentrations of oxaloacetate (OAA). On the left, the data is fit to the classic Michaelis-Menten equation, and the determined values of kcat, Km, and kcat/Km are provided. The curve does not follow the data, so a modified equation using Hill coefficients is used to fit to the data, as shown to the right in the figure. This equation provides an exponent "n" to the substrate concentration and to Km in the Michaelis-Menten equation. The Km term is renamed to Khalf, but these would be equivalent if n=1.
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Steps to reproduce
Kinetic constants were determined through a UV-Visible kinetic assay of human cytoplasmic malate dehydrogenase on oxaloacetate (OAA) with NADH as a cosubstrate. Measurements of the absorbance changes were taken in triplicate at 340 nm over the course of 30 seconds. The assay was performed in 3000 μL of 100 mM potassium phosphate assay buffer with 0.11 mg/mL NADH (pH 7.4). The enzyme concentration in the assay was 8.25 nM. OAA was added in varying concentrations ranging from 4.8 μM to 300 μM. The slope of each of change in absorbance versus time (AU/minute) were converted to the observed rate constant (kobs, or more accurately, the initial velocity) using the concentration of the enzyme in the assay and the molar extinction coefficient (ε) of NADH (6220 M-1cm-1). The kobs was then plotted against the concentration of the substrate in the assay and the kinetics constants were then obtained from this data using Mathematica.