Nitroasparaginase

Reaction of l-asparaginase from Escherichia coli with tetranitromethane converted up to 25% of the tyrosine residues to nitrotyrosine. In addition, however, acrylamide gel electrophoresis indicated aggregation of the nitroenzyme into multiple higher molecular weight species. Up to 60% of the monomers of this tetrameric enzyme were covalently cross-linked as a result of the tetranitromethane treatment. Although extensive inactivation of asparaginase activity occurred, glutaminase activity intrinsic in the native enzyme and the hydrolysis of β-cyano-l-alanine were relatively more inactivated. The rate of covalent binding of 5-diazo-4-oxo-l-norvaline to the active site of the enzyme was increased 2-fold in the nitroenzyme. These studies suggest caution in the treatment of oligomeric enzymes with tetranitromethane since extensive cross-linkage as well as alterations of substrate specificity can occur.