Estimation of the kinetic parameters (Km & Vmax) and the optimization of yeast alcohol dehydrogenase (ADH) assay.

Abstract

Alcohol dehydrogenases (ADHs) are zinc-containing enzymes that catalyze the oxidation of alcohols to aldehydes or ketones. The enzymes also play a critical role in the metabolism of a number of drugs and metabolites containing alcohol functional groups. Kinetic parameters of yeast alcohol dehydrogenase (ADH) was estimated using spectrophotometer, optimum temperature and pH for ethanol, as well as ADH specificity to other substrates was determined. Results from the assay showed value for the initial velocity of reaction of the enzyme estimated as 0.297. Change in concentration of NADH which correspond to ADH activity for ethanol was calculated as 48nanokatals (4.8 X 10-5 mol-1l), the results also showed average values of the Km and Vmax of ADH for ethanol as estimated from Michaelis-Menten and Line weaver-Burk double reciprocal plots, to be 21.5mM and 0.426 respectively. Optimum temperature and pH of ADH for ethanol were estimated to be 25 °C and 8 respectively. Although, reaction specificity of ADH to other substrates showed gradation between propan-2-ol and ethanol with ethanol showing highest activity and methanol least. 2-propene-1-ol also presented high enzyme activity relative to ethanol. In most of enzyme assays, enzyme activity is determined by measuring the rate of conversion of substrate or rate of production of products within a given period of time. In this experiment, the rate of oxidation of NADH was monitored since NADH has a known maximum light absorbance at 340nm.