110 MICROSOMAL PARTICLES 



HO 



I || II 



- CH 2 -C-C - 0- P-O- Ribose -Adenine" 1 " 

 I l 



NH 3 + 0~ 



Fig. 1. Tryptophanyl adenylate. 



known to react promptly with hydroxylamine to yield the corresponding hy- 

 droxamates [1-4]. Furthermore, it has been reported that synthetic leucyl 

 adenylate formed adenosine triphosphate when incubated with inorganic pyro- 

 phosphate and an activating enzyme purified from Escherichia coli [8]; a simi- 

 lar experiment has been carried out with methionyl adenylate and an activat- 

 ing enzyme isolated from yeast [6]. An additional piece of evidence consistent 

 with the formation of an anhydride linkage between the phosphoric acid 

 group of adenylic acid and the carboxyl group of amino acids has been ob- 

 tained in experiments with amino acids labeled with O 18 ; transfer of O 18 from 

 the carboxyl group of the amino acid to adenylic acid was associated with 

 enzymatic activation. The pyrophosphate formed did not contain appreciable 

 quantities of O 18 [9]. 



The available data are consistent with the hypothesis that aminoacyl adenyl- 

 ates are intermediates in the amino acid activation reaction, but the formation 

 of such anhydrides has not yet been shown. Although there is as yet no experi- 

 mental demonstration of the net synthesis of acyl adenylates in acetate or fatty 

 acid oxidation, the respective enzyme systems are apparently able to utilize 

 added synthetic acyl adenylate derivatives [1-3]. Previous inability to detect 

 the formation of such intermediate anhydrides may be related to the instability 

 of the anhydrides and perhaps also to the high affinity of the enzyme for the 

 anhydride; accordingly the actual intermediate in these activation reactions 

 may be enzyme-bound acyl adenylate. 



We have attempted to obtain evidence for the net synthesis of tryptophanyl 

 adenylate by the tryptophan-activating enzyme of beef pancreas [7]. In these 

 studies, we have used aminoacyl adenylates prepared as described in the fol- 

 lowing paper by Castelfranco et al. [10]. Although tryptophanyl adenylate is 

 hydrolyzed rapidly at pH 7.2 and 37° C, we have found that only about 10 per 

 cent of the anhydride is hydrolyzed in 2 hours at pH 4.5 and 0°. Paper 

 ionophoresis at pH 4.5 in 0.05 M ammonium formate buffer at 0° indicated 

 that tryptophanyl adenylate was positively charged and moved with a greater 

 mobility than tryptophan itself (fig. 2) . The positively charged band quenched 

 the fluorescence of the paper under ultraviolet light and gave the ninhydrin 

 color reaction. Elution of this material from the paper strip yielded an alkali- 

 labile compound which formed a hydroxamic acid promptly on treatment with 

 hydroxylamine. The hydroxamate was identified as tryptophan hydroxamate 

 by paper chromatography in several solvent systems. Incubation of the eluted 

 compound with the tryptophan-activating enzyme, magnesium chloride, inor- 



