Studies on aspartase. IV. Reversible denaturation of Escherichia coli aspartase.
Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) of Escherichia coli, denatured in 4 M guanidine-HCl, was renatured in vitro by simple dilution with a concomitant restoration of the activity. While the native enzyme exhibited a marked negative Cotton effect centered at 233 +/- 1 nm in optical rotatory dispersion, the enzyme denatured in 4 M guanidine-HCl retained little optical activity. Upon dilution of the denatured enzyme, however, more than 90% of the ordered structure was recovered in 1 min, while the restoration of the activity proceeded much more slowly. Estimation of molecular weights by gel permeation chromatography indicated that the tetrameric enzyme is subject to reversible dissociation into monomeric subunits under the experimental conditions. Various environmental factors such as temperature, pH and protein concentration exhibited profound influence on the rate and extent of the reactivation. In order to examine the correlation between the restoration of the activity and the quaternary structure, electron microscopic inspection of the kinetic processes of reversible denaturation was attempted. Upon dilution of the denatured enzyme at 4 degrees C, neither the activity nor tetrameric images were detected over several min. Upon the temperature shift up to 25 degrees C, however, the activity regain was rapidly proceeded concomitant with the appearance of tetrameric molecules. These results are compatible with the possibility that the subunit assembly is an essential prerequisite, thought not sufficient, for enzyme activity.
D000642:Ammonia-Lyases / Q000378:metabolism*; D001220:Aspartate Ammonia-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D009905:Optical Rotatory Dispersion; D009994:Osmolar Concentration; D011489:Protein Denaturation; D013696:Temperature
D006146:Guanidines; D046911:Macromolecular Substances; D000642:Ammonia-Lyases; D001220:Aspartate Ammonia-Lyase; D008274:Magnesium