CHEMICAL PATHWAYS 97 



hydroxylamine, pantoylhydroxamic acid and inorganic pyrophosphate are 

 formed in stoichiometric amounts, but this reaction is much slower than 

 the exchange. These experimental facts can be explained by the following 

 scheme (Maas, 1955) — 



E + ATP ^-"-"- "^ E-AMP ~pantoate + PP 

 E-AMP ^pantoate + NH^OH ^ E + AMP + pantoyl-NHOH 



The formation of a bond between AMP and pantoate was confirmed by the 

 observation of a transfer of i^O between the carboxyl group of pantoic acid 

 and AMP. 



It was soon found that a relatively slow pyrophosphate-ATP exchange, 

 which takes place in soluble extracts of rat liver, is strongly stimulated by a 

 mixture of the natural amino acids, and that hydroxamates are formed in 

 the presence of amino acids and high concentration of hydroxylamine; 

 leucylhydroxamate was identified among them (Hoagland, 1955). Com- 

 pletely similar observations were made by De Moss and Novelli (1955) 

 with extracts of many bacterial strains as well as yeast and moulds. That 

 the reactions involved are quite similar to those of pantoate activation was 

 soon confirmed. Leucyladenylate prepared by chemical synthesis when 

 incubated with these bacterial extracts and inorganic pyrophosphate forms 

 ATP, and free leucine appears (De Moss et al, 1956). Heavy oxygen is 

 exchanged between the carboxyl of an amino acid and the AMP moiety of 

 ATP (Hoagland et al., 1957; Bernlohr and Webster, 1958) showing that 

 at some time during the process the carboxyl group of the amino acid 

 shares one oxygen atom with AMP, not with the pyrophosphate residue. 



These experiments clearly established the existence of a direct activation 

 of amino acids. Amino acid activation enzymes were then sought and found 

 in all kinds of animal or plant tissues and in micro-organisms (see reviews 

 by Novelli and De Moss, 1957, and by Chantrenne, 1960). 



The discoverers of the amino acid activation enzymes noticed that if the 

 total concentration of amino acids is kept constant, the ATP-pyrophos- 

 phate exchange is promoted as the number of individual amino acids is 

 increased. This strongly suggested that the different amino acids did not 

 compete for a common activation enzyme, and that there were probably 

 several enzymes with different specificities. When activation was studied 

 separately for each individual amino acid, great differences in efficiency in 

 promoting the pyrophosphate exchange were observed between them (De 

 Moss and NovelH, 1955, 1956; Hoagland et al, 1956; Berg, 1956). Some of 

 the natural amino acids had so little effect on the exchange that for a time it 

 was believed that a few of the natural amino acids only can be activated in 

 such reactions. Later on, activation of all amino acids was observed in 

 bacteria (Nisman et al., 1957, 1958; Nisman, 1959) in animal and plant 

 tissues (Lipmann, 1958; Webster, 1959). But it remains that the reaction 



