96 THE BIOSYNTHESIS OF PROTEINS 



result in polypeptide formation. However attractive this hypothesis may 

 have looked for some time, no convincing evidence for the operation of such 

 a mechanism has been provided, and direct attempts to test the idea of the 

 participation of glutathione for instance as an initiator of amino acid 

 incorporation have led to a negative conclusion (Hendler and Greenberg, 

 1954; Flavin and Anfinsen, 1954; Askonas et ah, 1955; Barry, 1956). 

 Besides, glutamyl transpeptidases are found only in a very few tissues 

 (Revel and Ball, 1959). If polypeptides were formed by a series of trans- 

 peptidations at the energy level of the peptide bond, the concentration of 

 free intermediary peptides would be rather high, for the equilibrium 

 constant of such transpeptidations should be close to unity. And no free 

 peptides have been found to accumulate in living cell to any considerable 

 extent. There is at present no positive reason to think that proteolytic 

 enzymes play an important part in protein synthesis, neither that they play 

 any part in the process. It is not excluded, however, that they might be 

 involved in the finishing steps of protein formation (Horowitz and Hauro- 

 witz, 1959); limited proteolysis is clearly involved in zymogen activation 

 (Davie and Neurath, 1955; Desnuelle and Fabre, 1955). 



An alternative to the hypothesis considered above for explaining how 

 energy is funnelled into protein synthesis, is to assume that the amino 

 acids, or at least some of them, are activated directly in some process 

 involving ATP, and that the activated forms of the amino acids then con- 

 dense into polypeptides. 



B. THE ACTIVATION OF AMINO ACIDS 



1 . Amino Acid Activation Enzymes 



Analysis of the enzymic acetylation of aromatic amines by Lipmann's 

 group led to the basic discovery that ATP, which is used up stoichio- 

 metrically in the process, is not split in the 'usual' way into ADP and 

 phosphate; instead, it is broken into AMP and inorganic pyrophosphate 

 (Lipmann, 1952; Jones et al., 1953). This was the first observation of the 

 direct utilization of the second energy rich bond of ATP. Another example 

 of this new type of ATP cleavage was soon found by Maas and Novell! 

 (1953) in pantothenic acid synthesis from pantoic acid and ^-alanine. Ex- 

 periments with purified preparations of the enzyme provided evidence that 

 the formation of pantothenic acid involves the production of an enzyme- 

 bound pantoyl adenylate as an intermediate. The evidence was as follows : 

 the purified preparation catalyses an exchange of the two terminal phos- 

 phates of ATP with inorganic pyrophosphate, but this exchange requires 

 the presence of pantoate specifically. In the presence of concentrated 



