Intermediate Formation of Dipeptide-Phosphate Anhydride in Enzymatic Tripeptide Synthesis

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

“…It was previously postulated that nucleotide functions in a catalytic role and thus represents a portion of the enzyme-bound activated substrate complex; a tentative scheme for the reaction catalyzed by tripeptide synthetase was presented (Figure 4; Nishimura et al, 1964), which is analogous to that proposed for glutamine synthetase (Krishnaswamy et al, 1962). The present observations support the belief that the ADP formed in the reaction between enzyme, ATP, and dipeptide in the absence of acceptor remains attached to the enzyme.…”

“…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). These observations led to the conclusion that enzyme-bound 7-glutamyl-a-aminobutyryl phosphate was formed under these conditions, and experiments with chemically synthesized 7glutamyl-a-aminobutyryl phosphate have supported this interpretation (Nishimura et al, 1964).…”

“…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.…”

Flexible Loop That Is Novel Catalytic Machinery in a Ligase. Atomic Structure and Function of the Loopless Glutathione Synthetase

“…The catalytic mechanism of GSHase has been proposed to proceed via phosphorylation of the carboxylate of the cysteine moiety of -Glu-Cys to yield -L-glutamyl-L-cysteinyl phosphate (Meister, 1974). This intermediate is subsequently attacked by glycine, followed by loss of inorganic phosphate, leading to product formation (Nishimura et al, 1964). A similar intermediate, acyl phosphate, is also involved in the reaction of the other C-N bond ligases (Mullins et al, 1990;Meek & Villafranca, 1980;Bass et al, 1984).…”

Advances in the Chemistry of Carbodiimides

“…The products (CCXXIII) are probably formed by the nucleophilic attack of the amine on the phosphorus atom of the inter- f. Mixed Anhydrides Mixed anhydrides (CCXXIV) of amino acids and adenylic acids have been shown to be the "activated" forms through which amino acids are incorporated into proteins (257,487,827). A number of such anhydrides (e.g., luciferyl adenate (582) and L-methionyl adenate (CCXXIV, R = CH3SCH2CH2) (45, 46)) have been synthesized from the appropriate acids using dicyclohexylcarbodiimide as the condensing agent (45,386,428,450,468,519,575,582,597,871).…”