Activity of Liver Glutamine Transaminase toward l-γ-Glutamyl Hydrazones of α-Keto Acids

Abstract

Abstract Rat liver glutamine transaminase acts on γ-glutamyl hydrazones of α-keto acids to yield the corresponding l-amino acids and 3-hydroxy-tetrahydro-6-pyridazinone-3-carboxylic acid; the latter compound undergoes nonenzymatic dehydration in acid to yield 1,4,5,6-tetrahydro-6-pyridazinone-3-carboxylic acid. l-Amino acid oxidase (snake venom) also catalyzes the formation of 3-hydroxy-tetrahydro-6-pyridazinone-3-carboxylic acid (and ammonia) from γ-glutamyl hydrazide. In the transaminase-catalyzed reaction in which glycine is formed from the γ-glutamyl hydrazone of glyoxylate, no evidence was obtained for the formation of free glyoxylate during the reaction, nor did added free glyoxylate equilibrate with γ-glutamyl hydrazone-bound glyoxylate. The relative rates of reaction observed with a series of γ-glutamyl α-keto acid hydrazones were similar to those of the corresponding glutamine-α-keto acid transamination reactions; in general, the rates with the γ-glutamyl α-keto acid hydrazones were greater than those of the corresponding glutamine-α-keto acid transamination reactions. Possible reaction pathways for the enzymatic transformation of the γ-glutamyl α-keto acid hydrazones have been deduced. Albizziin (l-α-amino-β-ureidoproprionic) acid was found to effectively replace glutamine in the glutamine-α-keto acid transamination reaction; the high ultraviolet absorbance of the dehydrated product of the α-keto acid analog of albizziin was used as the basis for a highly sensitive assay for liver glutamine transaminase activity. Studies of the albizziinglyoxylate transamination reaction indicated that the enzyme exhibits ping-pong kinetics. The data suggest that the enzyme has binding sites for glutamine and α-keto acids which overlap. The action of the enzyme on γ-glutamyl-α-keto acid hydrazones appears to involve initial binding of the glutamyl moiety at the glutamine site of the enzyme followed by transformations which are associated with movement of the attached α-keto acid moiety into the active site. The preparation and properties of the l-γ-glutamyl hydrazones of a number of α-keto acids are described.