COENZYMES DERIVED FROM B VITAMINS 199 



to produce growth responses were definitely determined by the presence 

 or absence of purines, thymine, and the amino acid, methionine. 286, 287 



The hypothesis that these essential metabolites were products of 

 processes, one or more reactions of which involve p-aminobenzoic acid, 

 was greatly strengthened when quantitative concepts developed for 

 isolated enzyme systems were shown to be applicable also to the inhibition 

 of growth in bacteria. 288 It was deduced that a simple mathematical 

 analysis of the manner in which a compound counteracts inhibition of 

 bacterial growth indicates the role of this substance in the inhibited 

 system. That is, it is possible to determine whether a substance is (1) the 

 substrate (or precursor of the substrate) of the reaction, (2) a product 

 of the reaction (or derived from it), or (3) a part of the catalytic 

 mechanism of the reaction (Chapter ID). 



Whereas the antagonism between p-aminobenzoic acid and the sulfon- 

 amides is competitive, the concentrations of the pyrimiclines, purines, 

 and amino acids required to initiate growth in cultures which had been 

 completely inhibited by sulfonamides were found to be entirely inde- 

 pendent of the level of the inhibitor, thus substantiating the belief that 

 these compounds are "products" of p-aminobenzoic acid functioning, 289 

 and that this vitamin is required for their synthesis. What these synthetic 

 processes have in common, though, was not recognized until (1) tracer 

 studies had demonstrated the existence of the single carbon intermediate, 

 and (2) inhibition studies had established the type of reactions catalyzed 

 by p-aminobenzoic acid. 



The link connecting p-aminobenzoic acid with the metabolism of the 

 single carbon unit was disclosed when the chemical nature of a reactant 

 in a p-aminobenzoic acid catalyzed process was established. 290 A sub- 

 stance of unknown structure accumulated in the culture medium of 

 Escherichia coli whenever the growth of the organism was partially in- 

 hibited by sulfonamides. 291 The compound presumably piled up because, 

 after its formation, sulfonamides blocked the reaction by which it 

 normally was utilized. Hence it represented a compound related to the 

 substrate of a p-aminobenzoic acid-catalyzed reaction. The compound 

 which accumulated was shown to possess a molecular structure which 

 would make it a logical precursor of purines. It is an imidazole so sub- 

 stituted that if direct condensation with a single carbon unit were effected, 

 a naturally occurring purine hypoxanthine would be formed. It was 

 therefore postulated that one of the functions of p-aminobenzoic acid 

 was to catalyze a reaction in which the single carbon unit is introduced 

 into the 2 position of the purine nucleus and that one of the direct effects 

 of sulfonamide inhibition is the blocking of reactions in which the single 

 carbon unit participates. 290 



