Determination of the Kinetic Constants With Three Alcohol Substrates

The objective of this experiment was to compare the catalytic rate constant, kcat, the Michaelis constant, Km and the apparent second-order rate constant, kcat/ Km , for ethanol, propane-1-ol and butane-1-ol.The various reagents prepared and used in the experiment included the reaction buffer, 0.15 M sodium pyrophosphate with 1mM dithiothreitol, adjusted to pH 8.5 and room temperature. the three substrate solutions, ethanol (0.24M), propane-1-ol (0.24M) and butane-1-ol (0.24M) all adjusted to room temperature. coenzyme NAD+ solution (15mM). yeast alcohol dehydrogenase ( 58 U/mg. Sigma) prepared as a stock solution containing 0.1 mg protein/ml in distilled water and the protein content was accurately determined by measuring the absorbance at 280nm in comparison with a standard solution (1mg/ml) of yeast enolase having A280 value of 0.894. The stock solution of yeast ADH was suitably diluted with 10 mM potassium phosphate buffer, pH 7.4 containing 1 mg/mL of bovine serum albumin to produce A340 /10s of between 0.015 and 0.025 in the ADH assay with an ethanol concentration of 80 mM. The absorbance readings were taken using two 3-ml plastic cuvettes in a Novaspec spectrophotometer adjusted to 340nm. The instrument was adjusted to zero absorbance with a cuvette containing 3000 l of distilled water prior to taking the test readings.The assay mixture (total volume, 3000 μl) in the cuvette consisted of 1000μl 0.15 M sodium pyrophosphate buffer with mM dithiothreitol, pH 8. 1000 l of 0.24 M ethanol/propane-1-ol/butane-1-ol (yielding the equivalent of 0.08M ethanol in 3 ml of the assay mixture) or suitable dilutions thereof (yielding 0.04M, 0.02M, 0.01M and 0.005M alcohol substrate, Table 1). 100 l 15 mM NAD+ and the contents mixed well after covering the mouth of the cuvette with parafilm. Lastly, after wiping the optical surfaces of the cuvette, 100 l yeast alcohol dehydrogenase were added and the clock started.