Modification of catalytic groups in lysozyme with ethylenimine

Abstract

The reaction of lysozyme with ethylenimine has been studied at neutral and acidic pH. At least four singly modified lysozyme derivatives have been isolated. From the product analyses, only carboxyl groups in the protein were found to react. One of the derivatives, 1, was very labile and reverted back to native lysozyme during the isolation procedure. Its formation was markedly inhibited by the presence of tri(N-acetyl-D-glucosamine), indicating that the modified carboxyl residue in 1 is at or close to the binding site of lysozyme for the trisaccharide. Two other derivatives were identified as 2-aminoethyl esters of Glu-35 (2) and Asp-52 (3). At pH 10 and room temperature, these derivatives rearranged to the corresponding 2-hydroxyethylamide derivatives (5 and 6). A fourth derivative (4) did not contain an ethanolamine moiety; nevertheless, its Glu-35 was found to be modified by sequence analysis. Lysozyme derivatives modified at Glu-35 and Asp-52 were inactive toward glycol chitin but retained high affinity for tri(N-acetyl-D-glucosamine). Thus, Glu-35 and Asp-52 are essential for enzyme activity. A mechanism for the selective modification of these carboxyl residues in lysozyme has been proposed and related to the metal binding ability of lysozyme.

Chemical modification of carboxyl groups in glucoamylase from Aspergillus niger

“…Similar findings have been obtained previously with lysozyme. In this case, two different types of enzyme derivatives with singly modified catalytic carboxyl groups, Asp52 or Glu35 (5), could be identified (58). Evidence for a selective reaction of ethylenimine with essential carboxyl groups has thus been demonstrated for two carbohydrases, a behaviour perhaps promoted by the basic character of the compound.…”

“…Ethylenimine modifications were carried out by treatment of glucoamylase G 1 ( 1 -50 mg. ml-~) in water at pH 5.7 with 20-1400-fold molar B SVENSSON ct ill.; Essential carboxyl groups in glucoamylase excess of reagent for 24 h essentially as earlier described (58). Excess reagent was removed by dialysis prior to determination of the extent of modification and remaining enzymic activity (see 2.2.2).…”

Antivirals against animal viruses

“…tyrosines 20 and 23 were nitrated to nitrotyrosins or these reduced to aminotyrosins and the 6 tryptophans in turn were modified by reaction with 2-nitrophenyl sulfenyl chloride [114,115]), and in 1971 new lysozyme derivatives were generated by modifying its lysine residues by either guanidination, acetylation, succinylation, or maleylation [116,117]. Other simple lysozyme modifications include addition of fluorescein-isothiocyanate [118], chemical conversion of aspartic acid 52, a catalytic residue in hen egg-white lysozyme, to homoserine [119], ozone oxidation of tryptophan 62 to N 0formylkynurenine [120,121], selective modification of the aspartic acid at position101 in lysozyme by carbodiimide reaction [122], and modification of the enzyme's catalytic groups with ethylenimine [123]. All this research into lysozyme derivatives finally led into more radical and permanent modifications of the protein, such as the ADP-ribosylation of lysozyme described by Skórko and Kur [124].…”

Aminoalkylaziridines as substrates and inhibitors of lysyl oxidase specific inactivation of the enzyme by n- 5-aminopentyl aziridine

“…N-(4-Aminobutyl)aziridine, a potent inhibitor of cellular polyamine uptake, irreversibly inactivates diamine oxidase (9). These compounds are potentially covalent inactivators in view of their ability to alkylate an enzyme nucleophile at the aziridinyl moiety and to form Schiff base adducts with the carbonyl cofactor at the primary amino group (12)(13)(14)(15). Thus, a series of aminoalkylaziridines have been assessed as novel active site directed inhibitors of lysyl oxidase in the present report.…”