Enzymatic Transfer of the Gamma-glutamyl Group between Naturally Occurring Aniline and Phenylhydrazine Derivatives in the Genus Agaricus.

This report describes an unusual substrate specificity associated with the y-glutamyltransferase present in sporophores of Agaricus bisporus, the mushroom of commerce in the United States. A study of the properties of such an enzyme from this organism was prompted by recent reports of the isolation and characterization of two unique 7-glutamyl derivatives, N(y-L(+)-glutamyl)-p-hydroxyaniline 1 and fl-N(7-L(÷)glutamyl)-p-hydroxymethylphenylhydrazine (agaritine)2, 3 from fruiting bodies of species of Agaricus ~. Recognition of an enzyme in soluble extracts of A. bisporus that catalyzed the hydrolysis of the hydrazide linkage of agaritine 2 led us to investigate the possibility that such a reaction might reflect the functioning of an enzymatic process for the transfer of y-glutamyl residues to other acceptors in addition to water. Protein preparations from A. bisporus, purified for hydrolase activity about 26-fold by DEAE-cellulose column chromatography and precipitation with (NH4)~S04, catalyzed the synthesis of y-glutamylhydroxamic acid when incubated with z-glutamine and hydroxylamine. Hydrazine, a strictly competitive inhibitor of hydroxylamine in this process, also served as an acceptor of the y-glutamyl group. No evidence could be obtained for the participation of adenine nucleotides or metal ions in the reaction 4-6 nor for transfer of the y-glutamyl group to amino acids 7.n. More rapid and extensive transfer was observed when donors were used that were more effective than glutamine as substrates for hydrolytic cleavage. Among such donors were the 7-L-glutamyl derivatives of cyclohexylamine, p-hydroxyaniline, and phenylhydrazine (or p-hydroxymethylphenylhydrazine). This and several other lines of evidence (e.g. constancy of ratios of specific activities toward hydrolysis and transfer throughout the purification procedure, identity of rates of heat and pH inactivation, inhibition of hydrolysis by nitrogenous 7-glutamyl acceptors), have lent support to the conclusion that hydrolytic and glutamyl-transfer reactions are catalyzed by the same protein. Evidence for several of the reactions catalyzed by the mushroom glutamyltransferase is given in diagrammatic form by the chromatographic patterns shown in Fig. I. When NH4+, at rather high levels, was the acceptor, glutamine was formed in the presence of a donor such as v-glutamylcyclohexylamine (Expt. A) or agaritine. Both glutamine and 7-glutamyl-p-hydroxyaniline served as donors of the glutamyl group to phenylhydrazine** (Expts. B and C, respectively). Finally, in a situation essentially the reverse of that in Expt. C, agaritine functioned as a y-glutamyl donor to p-hydroxyaniline (Expt. D).