Modification of three enzymes of the β-ketoadipate pathway by N-methyl-N′-nitro-N-mtrosoguanidine

The sensitivities of three enzymes of the β-ketoadipate pathway to inactivation by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) were determined in vivo and in vitro under conditions compatible with mutagenesis.One enzyme, β-ketoadipate enol-lactone hydrolase, is very sensitive to inactivation by low concentrations of MNNG. This enzyme is also sensitive to inactivation by N-ethylmaleimide and mercurial reagents. The free sulfhydryl content of native enol-lactone hydrolase was determined to be two moles free sulfhydryl per mole of enzyme. A 95% inactivation of enol-lactone hydrolase by MNNG results in a masking of slightly more than one mole sulfhydryl per mole enzyme.Muconate lactonizing enzyme is moderately sensitive to inactivation by low concentrations of MNNG, but is not inactivated by sulfhydryl reagents. Muconolactone isomerase is resistant to inactivation by low concentrations of MNNG and is not inactivated by sulfhydryl reagents. Upon exposure to high concentrations of MNNG, muconolactone isomerase is rapidly inactivated. Spectrophotometric evidence indicates the lysine residues are nitroguanidinated proportionally with a loss in the enzymatic activity.These data indicate that the exposure of cells to low concentrations of MNNG should affect the activity of enzymes with essential sulfhydryl groups.