Amino acid residues in asparaginase ( Escherichia coli Hap) associated with its enzymic activity

Abstract

The relationship of the amino groups and tyrosine, arginine, tryptophan, serine and cysteine residues in asparaginase ( l-asparagine amidohydrolase, EC 3.5.1.1) isolated from Escherichia coli HAP with the enzymic activity was investigated by measuring the reactivity of each residue or group with various chemicals, together with the change of enzymic activity. The following facts were revealed: The total 90 amino groups in the asparaginase molecule were classified into three types; (1) approximately 32 residues reactive with monochlorotrifluoro-p- quinone (CFQ), (2) approximately 45 residues becoming reactive with CFQ after alkali denaturation and (3) the remaining 13 residues nonreactive with CFQ. 4 ± 0.5 amino groups reactive with CFQ in the native enzyme were closely associated with the enzymic action. About 15 out of 47 tyrosine residues in the enzyme molecule were modified with tetranitromethane and the activity of the modified molecule decreased to 8% of the original level. About 12 out of the total 32 arginine residues and 2 or 3 out of the total 5 tryptophan residues per molecule were modified with glyoxal and H 2O 2-dioxane, respectively, and the activities of the modified enzymes decreased to 25% and 40% of the original. These activity drops may be attributed to a destruction of the tertiary structure of the enzyme. Serine residue reactive with diisopropyl-fluorophosphate (DFP) and cystein were not present in the asparaginase molecule. The subunit structure of the asparaginase molecule was discussed in relation to these data.