Specificity of γ-Glutamyl Cyclotransferase

Using the phthaloyl method, 18 γ-L-glutamyl peptides labelled with 14C in the N-terminal position have been synthesized. The products were isolated by simple procedures using a Dowex-1 column or high voltage electrophoresis. The synthetic peptides contain minor impurities of the corresponding D-glutamyl isomers. The proportion of D-isomer was determined by the use of glutamic decarboxylase, or by a new method using digestion with purified γ-glutamyl cyclotransferase and determination of the resulting 2-pyrrolidone-5-carboxylic acid (5-oxoproline). Evidence was obtained that γ-glutamyl cyclotransferase acts only on the L-form of γ-glutamyl substrates; the enzyme could, therefore, be used for preparation of γ-D-glutamyl peptides from their racemic mixtures. The specificity of γ-glutamyl cyclotransferase has been examined using pure enzyme prepared from pig liver, and extracts from tissues of rat and man. The basic structural requirement in substrates may be represented as γ-L-glutamyl—NH—CHR—COOH. The amino acid linked to the γ-glutamyl group must be in the L configuration.