Glycerol as an enzyme-stabilizing agent: effects on aldehyde dehydrogenase.
The potassium-dependent aldehyde dehydrogenase (EC 1.2.1.3), from yeast is markedly altered by the addition of high concentrations of glycerol or other polyhydric alcohols to aqueous buffers. Several lines of evidence suggest that the three-dimensional structure near the active site is involved: (i) The stability of the enzyme when stored at 2 degrees , or when subjected to repeated freezing and thawing, depends upon the presence of at least 30% (v/v) glycerol. (ii) In the same solvent, the K(m) value for DPN and the binding constant for benzaldehyde decrease by 3- and 10-fold, respectively, compared with the values obtained for these substrates in fully aqueous media. (iii) Competitive inhibition by trivalent arsenicals with respect to DPN is no longer observed in glycerol; the inhibition becomes mixed and the K(i) values increase by 5- and 50-fold, respectively, with arsenite and Mapharsen. (iv) Essential sulfhydryl groups, which are easily carboxymethylated in aqueous buffers, are not readily available in either glycerol or mannitol. The data are consistent with a change in topography induced by polyhydric alcohols in which sulfhydryl groups near the DPN-binding site are displaced to a more protected environment, where their reactivity is reduced. Since the stabilizing effects of such alcohols are frequently encountered, these results may have application to other enzymes.
D000447:Aldehydes; D000478:Alkylation; D001151:Arsenic; D001152:Arsenicals; D001665:Binding Sites; D002247:Carbon Isotopes; D004355:Drug Stability; D004789:Enzyme Activation; D005615:Freezing; D005990:Glycerol; D006018:Glycols; D007461:Iodoacetates; D007700:Kinetics; D008353:Mannitol; D009243:NAD; D010088:Oxidoreductases / Q000037:antagonists & inhibitors; D011188:Potassium; D011487:Protein Conformation; D012440:Saccharomyces / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013395:Sucrose; D013438:Sulfhydryl Compounds
D000447:Aldehydes; D001152:Arsenicals; D002247:Carbon Isotopes; D006018:Glycols; D007461:Iodoacetates; D013438:Sulfhydryl Compounds; D009243:NAD; D008353:Mannitol; D013395:Sucrose; D010088:Oxidoreductases; D001151:Arsenic; D005990:Glycerol; D011188:Potassium