L-Asparaginase from Erwinia carotovora

The physicochemical properties of the antileukemic Lasparaginase from Erwinia carotovora have been studied and compared with those of the corresponding clinically effective enzyme from Escherichia coli B. Although both enzymes are tetramers with similar molecular weight, the amino acid compositions are distinctly different and the Erwinia enzyme is more basic as judged by polyacrylamide electrophoresis. Of the 48 to 50 tyrosyl residues in native Erwinia asparaginase, only about 10% ionize with a normal pK, while 30% of these residues in the E. coli enzyme titrate normally. The native tetramer (s~(,,~ = 7.2) is considerably more stable than the corresponding tetramer from E. coli. Asparaginase from Erwinia is only partially dissociated in 8 M urea, whereas the E. coli enzyme is completely converted to the 1.7 S monomer under the same conditions. Guanidinium chloride at concentrations of 3.5 M completely dissociates Erwinia asparaginase. Dissociation is accompanied by the appearance of an ultraviolet difference spectrum with a maximum at 288 nm. The rate of dissociation is markedly increased by the addition of alcohols to the denaturant. Dissociation in the presence of alcohols occurs in two distinct steps whose rates increase as the length of the alkyl side chain of the alcohol increases. Succinylation of the lysyl residues results in marked increases in mobility of the modified enzyme on polyacrylamide gels, but does not affect the state of aggregation of the tetramer or its catalytic activity. In addition succinylation does not affect either the rate of dissociation in guanidinium chloride or the extent of reconstitution to the active enzyme. The presence of five bands on polyacrylamide gel electrophoresis of hybrids prepared from the native and succinylated enzyme indicates that Erwinia carotovora asparaginase is composed of four identical subunits.