Preparation of active hybrid enzymes composed of the native and chemically inactivated aspartase subunits from Escherichia coli.
The hybridization of the native and chemically inactivated aspartase from Escherichia coli was studied. Preparations of the tetrameric enzyme obtained by mixing the native and N-ethylmaleimide (NEM)-inactivated aspartase in 4 M guanidine-HCl followed by 51-fold dilution at room temperature retained catalytic activity. Affinity chromatography on AF-Red TOYO-PEARL separated several active components in the hybridized preparations, and the presence of [14C]NEM-inactivated subunits in the active hybrids was demonstrated. The addition of the native aspartase to Sepharose-bound NEM-inactivated enzyme in 4 M guanidine-HCl resulted in the formation of an immobilized enzyme with enzyme activity. The specific activity of the various hybrids, composed of unmodified and [14C]NEM-inactivated subunits, was roughly proportional to the number of unmodified subunits in each tetramer. Furthermore, when reversible denaturation was conducted on mixtures of the native and NEM-inactivated enzyme at various proportions, the enzyme activity recovered was proportional to the amount of the native enzyme added. These results strongly suggest that each subunit makes an independent contribution to the overall enzyme activity regardless of the presence of other subunits in the same molecule. The theoretical and practical implications of this work are discussed.
Imaishi
H
H
Department of Chemistry, Faculty of Science, Kyoto University, Japan.
Yumoto
N
N
Tokushige
M
M
D000642:Ammonia-Lyases / Q000378:metabolism*; D001220:Aspartate Ammonia-Lyase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002846:Chromatography, Affinity; D004800:Enzymes, Immobilized; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D011489:Protein Denaturation; D055503:Protein Multimerization
D004800:Enzymes, Immobilized; D006146:Guanidines; D046911:Macromolecular Substances; D000642:Ammonia-Lyases; D001220:Aspartate Ammonia-Lyase; D019791:Guanidine; D005033:Ethylmaleimide