L-Asparaginase from escherichia coli B. Succinylation and subunit interactions.

Asparaginase from Esckerickio coli B has been modified with increasing amounts of [r4C]succinic anhydride. Enzyme activity is enhanced when 15 % and 25 % of the lysyl residues are succinylated. Up to 40% of the lysyl residues were succinylated without destroying enzyme activity and without causing dissociation of this oligomeric protein. More extensive succinylation causes dissociation into subunits which exposes further sites for reaction with the cyclic anhydride. The number of succinyl groups covalently bound far exceeds the content of lysine groups, indicating that although succinic anhydride prefers to react with the e-amino group of lysyl residues it will also react with the functional groups of other amino acids. The results suggest that asparaginase contains two distinct classes of lysyl residues. The first 40% react readily with succinic anhydride and are not essential for catalytic activity; nor are they involved in subunit interactions. The remaining 60% are much less reactive and become available to the reagent only after the subunit has dissociated and functional groups which were hidden or were involved in the association of subunits are now exposed. Succinyl asparaginase dissociated more rapidly in the presence of 4.6 M urea than the native enzyme. The succinylated subunits can be reconstituted in part to a catalytically active tetramer. The fraction which does not fully reassociate has a sedimentation of 3.3 S, an emission maximum at 330 nm, compared with 317 nm for the tetramer or 345 nm for the monomer, and appears to be a dimer based on its behavior in a sucrose gradient. Hybridization of native and succinyl asparaginase produces three bands in addition to the starting proteins on polyacrylamide disc gel electrophoresis. These results constitute evidence that the native and modified enzymes are composed of four identical subunits.