With a PhD in biochemistry from Brooklyn Polytechnic Institute and an early job in the fermentation sciences department at Fleischmann’s, Owades probably knew more about fermentation and alcohol metabolism than perhaps any man who has ever lived.
The Thermodynamic Stability and Catalytic Activity of Yeast Alcohol Dehydrogenase at Different pH Values: an Undergraduate Biochemistry Investigation
Yeast alcohol dehydrogenase (YADH), which oxidizes ethanol to acetaldehyde according to Equation 1, has been a widely used enzyme for study in undergraduate biochemistry laboratory programs.~ CH3CH2OH+NAD + ----> CHBCHO +NADH+H + (1) YADH can be purchased from commercial sources, or it is easily isolated and purified from commercially available yeast. The enzyme's activity is conveniently measured spectro-photometrically and it is sufficiently stable for student use. In this article we report an experiment in which students first determine the enzyme's catalytic activity over a range of pH values and then use fluorescence spectroscopy to determine the thermodynamic stability of the YADH at the same pH values. Much to the student's surprise, they discover that YADH exhibits its maximal catalytic activity at pH values of 8.6-8.8 while it is thermodynamically most stable at pH 6.0.
Activity and Stability of Yeast Alcohol Dehydrogenase (YADH) Entrapped in Aerosol OT Reverse Micelles
The activity and stability of yeast alcohol dehydrogenase (YADH) entrapped in aerosol OT reverse micellar droplets have been investigated spectrophotometrically. Various physical parameters, e.g., water pool size, wo, pH, and temperature, were optimized for YADH in water/AOT/ isooctane reverse micelles. It was found that the enzyme exhibits maximum activity at wo = 28 and pH 8.1. It was more active in reverse rnicelles than in aqueous buffers at a particular temperature and was denatured at about 30°C in both the systems. At a particular temperature YADH entrapped in reverse micelles was less stable than when it was dissolved in aqueous buffer.
The Role of Zinc in Alcohol Dehydrogenase: V. THE EFFECT OF METAL-BINDING AGENTS ON THE STRUCTURE OF THE YEAST ALCOHOL DEHYDROGENASE MOLECULE
The role of zinc in the catalytic action of yeast alcohol dehydrogenase has been studied through the kinetics of the inhibition of activity by chelating agents (I), particularly l,lO-phenanthroline (2-4). In aqueous solution the complexes of 1, lo-phenanthroline with Zn++ ions exhibit characteristic absorption spectra, as do the complexes of this agent with the zinc atoms of yeast alcohol dehydrogenase and of other zinc metalloenzymes (5). the molecular stoichiometry of the enzyme-inhibitor complexes, deduced from spectrophotometric measurements, is in agreement with that inferred from kinetic data (6). Whereas the catalytic activity of the enzyme can thus be decreased by the localized attack of a chelating agent on a component of an “active site,” alterations in the protein structure may have similar functional consequences, of course. Chelating agents are here shown to induce changes in enzymatic activity both through such local action and through subsequent alterations of the macromolecular structure of yeast alcohol dehydrogenase. As a function of the time of exposure to chelating agents and of their concentration, the apoenzyme, molecular weight 151,000, dissociates into four equal subunits, molecular weight 36,000, while the four zinc atoms are removed concomitantly. Thus, a direct correlation between the enzymatic activity, the zinc content, and the protein structure of the enzyme can be shown to exist. A preliminary report has been made (7).
Effect of pH on the liver alcohol dehydrogenase reaction
New transient kinetic methods, which allow kinetics to be carried out under conditions of excess substrate, have been employed to investigate the kinetics of hydride transfer from NADH to aromatic aldehydes and from aromatic alcohols to NAD+ as a function of pH. The hydride transfer rate from 4-deuterio-NADH to beta-naphthaldehyde is nearly pH independent from pH 6.0 to pH 9.9; the isotope effect is also pH independent with kappa-H/kappaD congruent to 2.3. Likewise, the rate of oxidation of benzyl alcohol by NAD+ changes little with pH between pH 8.75 and pH 5.9; the isotope effect for this process is between 3.0 and 4.4. Earlier substituent effect studies on the reduction of aromatic aldehydes were consistent with electrophilic catalysis by either zinc or a protonic acid. The pH independence of hydride transfer is consistent with electrophilic catalysis by zinc since such catalysis by protonic acid (with a pK between 6.0 and 10.0) would show strong pH dependence. However, protonic acid catalysis cannot be excluded if the pKa of the acid catalyst in the ternary NADH-E-RCOH complex were smaller than 6.0 or smaller than 10.0. The two kinetic parameters changing significantly with pH are the kinetic binding constant for ternary complex formation with aromatic alcohol and the rate of dissociation of aromatic alcohols from enzyme. This is consistent with base-catalyzed removal of a proton from alcohol substrated and consequent acid catalysis of protonation of a zinc-alcoholate complex. The equilibrium constant for hydride transfer from benzaldehyde to benzyl alcohol at pH 8.75 is K-eq equals kappa-H/kappa-H equals 42; this constant has important consequences concerning subunit interactions during liver alcohol dehydrogenase catalysis.
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