86 MOLECULES, VIRUSES, AND BACTERLV 



growth, was responsible for blocking production of the intermediate; 

 also, the change in rate of production of the metabolite upon altering 

 the tryptophan concentration was immediate, showing the regulation 

 to be at the level of enzyme action rather than of enzyme synthesis. 



Similarly, in the purine biosynthetic pathway, purines blocked the 

 production by purine-requiring mutants of metabolic intermediates 

 such as 5-amino-4-imidazole-carboxamide (Gots, 1957). The end prod- 

 uct of the metabolic sequence must here also block an early step. Re- 

 cently such an inhibition has been demonstrated with a purified 

 enzyme. The earliest specific step in the pathway— the reaction of 

 phospho-ribosyl-pyrophosphate with glutamine to produce phospho- 

 ribosylamine— is strongly inhibited by various purine nucleoside di- and 

 tri-phosphate compounds which are a dozen steps removed from the 

 reaction ( Wyngaarden and Ashton, 1959 ) . 



Other feedback controls interact at a later stage of purine synthesis 

 in such a way as to partition inosinic acid between adenylic and guany- 

 lic acids (Magasanik, 1958). Guanylic-acid formation is regulated by 

 its ability to inhibit inosinic acid dehydrogenase, an enzyme required 

 for guanylic acid formation. ATP, a product of the alternative pathway, 

 is required for the production of guanylic acid, and conversely, in the 

 other branch of the pathway, GTP is required for the formation of 

 adenylic acid from inosinic acid. These and similar controlling factors 

 provide a most elegant explanation of how, in this case, feedback per- 

 mits partitioning of a metabolite between alternative pathways. 



Feedback inhibition in vivo and in vitro has been demonstrated in 

 the pyrimidine pathway (Yates and Pardee, 1956). In vivo, the pyri- 

 midine uracil was found to block the formation of orotic acid by pyri- 

 midine-requiring mutants of A. aerogenes (Brooke et al., 1954) or of 

 carbamyl aspartate in E. coli (Yates and Pardee, 1956). As Figure 5 

 shows, production of orotic acid by a mutant of E. coli was negligible 

 until the bacteria had utilized the available uracil, whereupon the 

 metabolite was rapidly produced. Inhibition was shown for aspartate 

 transcarbamylase, the first enzyme of the pathway; its activity could 

 be inhibited in whole cells and in broken-cell preparations. With the 

 latter, inhibition was obtained with various cytidine compounds but 

 not with uracil derivatives. More recent studies of this feedback inhi- 

 bition have been performed with the highly purified enzyme ( Gerhart 

 and Pardee, 1961). Cytidine triphosphate was the strongest inhibitor 

 ( Table I ) . The strength of inhibition depended on the structures of all 

 three parts of the molecule (base, sugar, and phosphate). These data 

 suggest that a part of the enzyme molecule comparable in size to the 

 entire inhibitor binds the latter. Since the inhibitor competes with the 

 much smaller substrate ( aspartic acid ) , the enzyme seems to have sites 

 to receive the inhibitor which are independent of the sites designed to 



