Bovine hepatic gamma-glutamyl hydrolase (conjugase) has been purified to homogeneity. A feature of the purification procedure was the use of high affinity macromolecular polyanion enzyme inhibitors which formed tight complexes with the enzyme altering its solubility, gel filtration, and ion exchange properties. The enzyme, which cleaves the gamma-glutamyl bonds of pteroylpolyglutamates, has a molecular weight of 108,000. It is a glycoprotein with an acid pH optimum, properties consistent with

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... s lysosomal localization. Zinc is essential for enzyme stability. The presence of highly reactive sulfhydryl groups was evident from the extreme sensitivity to oxidizing agents and organomercurials. Very little thermal denaturation occurs below 65 degrees, but the enzyme is extremely sensitive to 0uffer anions, in keeping with the polyanionic nature of the substrate. In order to study the mechanism of action of the enzyme, a wide range of pteroylpolyglutamates, N-t-Boc polyglutamates and free polyglutamates were synthesized containing L-[U-14C]glutamic acid residues in different positions. Two pteroyltriglutamate derivatives were also synthesized in which an alpha bond replaced one of the two available gamma bonds. Time course studies of the products of the action of conjugase on these various substrates enabled us to draw the following conclusions about the enzyme: (a) peptide bond cleavage occurred only at gamma-glutamyl bonds and the presence of a COOH-terminal gamma bond was essential for enzyme action; (b) bond cleavage occurred with equal facility at internal points of the peptide chain and the enzyme should therefore be more appropriately classified as an acid hydrolase; (c) longer chain gamma-glutamyl peptides were preferentially attacked by the enzyme, the cleavage of diglutamyl peptides being extremely slow; and (d) cleavage of gamma bonds was independent of the NH2-terminal pteroyl moiety. Studies with polyanions such as the glycosaminoglycans and dextran sulfate supported the concept that the polyanion structure of the substrate was a major factor in substrate-active site interaction.