Studies on aspartase. II. Role of sulfhydryl groups in aspartase from Escherichia coli.
Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli W contains 38 half-cystine residues per tetrameric enzyme molecule. Two sulfhydryl groups were modified with N-ethylmaleimide or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) per subunit, while 8.3 sulfhydryl groups were titrated with p-mercuribenzoic acid. In the presence of 4 M guanidine - HCl, 8.6 sulfhydryl groups reacted with DTNB per subunit. Aspartase was inactivated by various sulfhydryl reagents following pseudo-first-order kinetics. Upon modification of one sulfhydryl group per subunit with N-Ethylmaleimide, 85% of the original activity was lost; a complete inactivation was attained concomitant with the modification of two sulfhydryl groups. These results indicate that one or two sulfhydryl groups are essential for enzyme activity. L-Aspartate and DL-erythro-beta-hydroxyaspartate markedly protected the enzyme against N-ethylmaleimide-inactivation. Only the compounds having an amino group at the alpha-position exhibited protection, indicating that the amino group of the substrate contributes to the protection of sulfhydryl groups of the enzyme. Examination of enzymatic properties after N-ethylmaleimide modification revealed that 5-fold increase in the Km value for L-aspartate and a shift of the optimum pH for the activity towards acidic pH were brought about by the modification, while neither dissociation into subunits nor aggregation occurred. These results indicate that the influence of the sulfhydryl group modification is restricted to the active site or its vicinity of the enzyme.
D000642:Ammonia-Lyases / Q000378:metabolism*; D001220:Aspartate Ammonia-Lyase / Q000032:analysis / Q000378:metabolism*; D001224:Aspartic Acid / Q000031:analogs & derivatives; D001665:Binding Sites; D003553:Cystine / Q000032:analysis*; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008626:Mercuribenzoates / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation
D008626:Mercuribenzoates; D001224:Aspartic Acid; D003553:Cystine; D004228:Dithionitrobenzoic Acid; D000642:Ammonia-Lyases; D001220:Aspartate Ammonia-Lyase; D005033:Ethylmaleimide