Alcohol dehydrogenase (ADH) plays an important role in the anaerobic fermentation of yeast. This reports aims to analyse the kinetic parameters of ADH through spectrophotometry of ADH-catalysed reaction where ethanol is used as a substrate. The Lineweaver-Burk and the Eadie-Hofstee plots are used to linearly transform the hyperbolic form of the Michaelis-Menton equation and to calculate the accurate values of the kinetic parameters under consideration.
This results obtained from these plots and equation help tp determine the importance of Km values of enzymes and various factors affecting it such as pH, temperature, presence of metalloenzymes. A brief discussion about the poor substrate specificity of ADH towards ethylene glycol and methods to prevent the occurrence of acidosis in human being due to the presence of ethylene glycol is also presented. INTRODUCTION
Dehydrogenases enzymes oxidize a substrate by transferring hydrogen to an acceptor. (Branden et al. , 1975). Alcohol dehydrogenase (ADH- EC 1. 1. 1. 1) belongs to this group and catalyses many enzyme reactions (Sund and Theorell, 1963). Saccharomyces cerevisiae (Yeast) has three isoenzymes of ADH namely YADH-1, YADH-2 and YADH-3. YADH-1, which is important for fermentation, consists of four identical subunits, each containing a co-enzyme binding site and a bound zinc atom (Leskovac et al.
2002). Anaerobic conversion of Saccharomyces cerevisiae involves conversion of pyruvate (formed during glycolysis) into ethanal (acetaldehyde) in the presence of enzyme pyruvate decarboxylase (first step) and then reduction of acetaldehyde in the presence of ADH using co-enzyme NADH into ethanol, carbon dioxide and NAD+ (second step). The second step is reversible and these post-glycolysis reactions take place in the cytosol (Petro, 2005).
The above-mentioned reactions were the basis of this practical where the kinetics of ADH was closely monitored by spectrophotometric analysis. NADH has an absorption maximum at 340nm while the oxidized form has no absorption at this wavelength. A backwards reaction was carried out and an expected increase in absorbance of the solution was observed as at 340 nm as NADH was reformed (Suzuki et al. 2000). The role of kinetic parameters, maximal velocity (Vmax) and the Michaelis constant (Km) of ADH were also investigated.
The isoezyme YADH-2, which differs from YADH 1 at position 294 (methionine inYADH-1, leucine in YADH-2) is responsible for promoting the backward reaction by oxidizing ethanol to acetaldehyde. The higher activity of YADH-2 can be attributed to tighter binding of the longer chain alcohols and more rapid hydrogen transfer (Gould and Plapp, 1990). This background helps to define a hypothesis for this practical.