Asparaginase

Large Cotton effects associated with the π → π* and n → π* transitions of asparaginase substrates have been shown in substrates containing a 2nd asymmetrical carbon atom at the β position, diaminosuccinic acid monoamide and β-methylasparagine. Hydrolysis of the amide bond is accompanied by large changes in the rotatory strength of these transitions. These substrates have four possible isomers, l-l, d-d, l-d, and d-l. From the signs of the Cotton effects appearing on hydrolysis of mixtures of these isomers it can be determined which isomer is hydrolyzed. The enzyme has an almost absolute stereospecificity for the l-d isomer which defines the three-dimensional arrangement of the protein groups interacting with the substituents on the α carbon and the catalytic groups adjacent to the β-amide within rather narrow limits. While the l-l isomer of diaminosuccinic acid monoamide is not detectably hydrolyzed, the l-l isomer of β-methylasparagine is attacked at a rate 0.2 to 0.3% of that observed for l-asparagine, suggesting that a β-CH3 group can be accommodated in this configuration while a β-NH2 cannot.