Evidence for an Intermediate in the Synthesis of γ-Glutamyl-α-aminobutyrylglycine by Glutathione Synthetase

Tripeptide (Glutathione) Synthetase. Purification, Properties, and Mechanism of Action*

“…tides had equilibrated. In controls in which both labeled and unlabeled dipeptides were added initially together there was, as expected, relatively little formation of 14C-dipeptide hydroxamate (Nishimura et al, 1963). Subsequently, a compound was isolated from reaction mixtures containing enzyme, ATP, and yglutamyl-a-aminobutyrate, that exhibited the properties expected of 7-glutamyl-a-aminobutyryl phosphate (Nishimura et al, 1964).…”

“…Other Methods. 7-Glutamyl-a-aminobutyryl-1JC-hydroxamate was determined as previously described (Nishimura et al, 1963). Phenol extraction of the enzyme was carried out as described by Bieber et al (1964).…”

“…Studies on the Intermediate. Experiments of the pulselabeling type were reported previously (Nishimura et al, 1963) in which relatively large amounts of the enzyme were incubated with 14C-dipeptide and ATP; after brief incubation a mixture of hydroxylamine and an excess of unlabeled dipeptide were added; under these conditions, there was a much greater formation of 7-glutamyl-a-aminobutyryl-14C-hydroxamate than would be expected if the labeled and unlabeled dipep- " The standard assay system for hydroxamate formation was used containing 2.5 jimoles of dipeptide and 1.0 unit of enzyme (see the text). b Contained about equal amounts of the a-and /3-aspartyl peptides (5.0 junoles of this dipeptide was used).…”

“…1064) were obtained from Pharmacia and Brown Co., respectively. L-y-Glutamyl-L-a-aminobutyric acid was prepared from the unlabeled amino acids or from uniformly labeled L-glutamate-l4C as previously described (Nishimura et al, 1963(Nishimura et al, , 1964 and also by enzymatic transpeptidation using purified hog kidney 7-glutamyl transpeptidase (Orlowski and Meister, 1965;Mooz, 1967). 7-L-Glutamylcysteine (Strumeyer and Bloch, 1962) and the 7-glutamyl peptides listed in Table III (Otani and Meister, 1957) were prepared as described.…”

Evidence for Succinyl Phosphate as an Enzyme-bound Intermediate in the Reaction Catalyzed by Succinyl Coenzyme A Synthetase*

“…When the enzyme was incubated with [ -32 ] Q W_/everal recent investigations have dealt with the mechanism of the reaction catalyzed by succinyl coenzyme A synthetase: succinyl CoA + P¡ + ADP ^ATP + succinate + CoA Hager (1962), in an excellent review of this subject, has summarized the available evidence for and against a mechanism involving (1) enzyme-bound succinyl phosphate, (2) enzyme-bound phosphoryl coenzyme A, and (3) no discrete intermediates. The present studies were stimulated by earlier work in this laboratory on tripeptide synthetase in which direct evidence for the intermediate formation of an enzyme-bound dipeptide-phosphate anhydride was achieved (Nishimura et al, 1963(Nishimura et al, , 1964, and on glutamine synthetase in which data giving strong support for an enzyme-bound glutamyl phosphate intermediate were obtained (Krishnaswamy et al, 1962;Khedouri et al, 1964). In both of these reactions (Boyer et al, 1956;Kowalsky et al, 1956;Strumeyer, 1959) and in the reaction catalyzed by succinyl coenzyme A synthetase (Hager, 1962) it has been shown that 180 is transferred from inorganic phosphate to a carboxyl group or vice versa.…”