172 S. S. COHEN 



4. The syntliesis of the tripeptide glutathione, involves a stepwise forma- 

 tion of dipeptide and tripej)tide, as follows: 



K+ 



glutamate + cysteine + ATP > y-glutamyl cysteine + ADP + P (1) 



Mg++ 



K + 



y-glutamyl cysteine + glycine + ATP > glutathione + ADP -f P (2) 



Mg+ + 



In each reaction, the first step is postulated to be: 



E + ATP ^ E-P + ADP, followed in reaction 1 by (1) 



E-P + glutamate ^ E-y-glutamyl + P (2) 



E-y-glutamyl + cysteine ^ E + y-glutamyl-cysteine (3) 



Thus, four different courses have been observed for the four model systems 

 studied. Only one was found to involve coenzyme A. In two instances, the 

 cleavage of ATP yielded ADP + P; in two others, AMP + PP were formed. 

 Nevertheless, the carboxyl group, later to be incorporated into the amide 

 bond, was activated in each case. 



Of the four systems studied, that of carboxyl activation in pantothenate 

 synthesis has been suggested most often to bear the closest similarity to the 

 activation of amino acids, since the latter also involves the hberation of PP 

 from ATP, as mentioned in an earher section. The case of pantoate activa- 

 tion is also superficially analogous to the activation of other acyl moieties 

 (acetate, and a number of other groups, sulfate, carbonate, etc.), although a 

 pantoyl adenylate has not yet been described. As noted earlier, acetate 

 catalyzes the exchange of pyrophosphate with ATP in yeast extracts with 

 the intermediate formation of adenyl acetate, which then reacts with coA 

 to form acetyl-ScoA (Berg, 1955, 1956a). A comparable sequence and role is 

 recognized for butyrate (Talbert and Hueimekens, 1956) and higher fatty 

 acids (Jencks and Lij^mann, 1957). 



In the latter case, the same enzyme system from hver capable of activat- 

 ing fatty acids also was able to activate one amino acid, phenylalanine. The 

 formation of the j)hosj)hocarboxyanhydride of the amino acid was indicated 

 both by the release of PP and by a reactivity with hydroxylamine to form a 

 characteristic hydroxamate, useful in the detection and estimation of this 

 class of compounds. In this study the existence of an acyl adenylate deacyl- 

 ase was also detected. 



The role of sulfate in pyrophosphate exchange with ATP to form adenyl 

 sulfate, has been described by Hilz and Lipmann (1955). This compomid is 

 an intermediate in the formation of adenosine-3'-,phospho-5'-phosphosulfate, 

 the coenzyme for the transfer of sulfate in the synthesis of molecules hke 

 chondroitin sulfate (D'Abramo and Lipmami, 1957) and phenol sulfate 

 (Robbins and Lipmann, 1957). 



