The enzymic synthesis of chorismic and prephenic acids from 3-enolpyruvylshikimic acid 5-phosphate.

The conversion of 3-enolpyruvylshikimate .5-phosphate (I) to phenylpyruvate was studied in extracts of Escherichia coli. A separation and partial purification was achieved of the two enzymic activities concerned: chorismate synthetase which catalyzed the 1,4-conjugate elimination (E2’) of orthophosphate from I to yield chorismic acid (II), and the enzyme complex catalyzing the reactions chorismate + prephenate (III) -+ phenylpyruvate. Chorismate synthetase was inactive under aerobic conditions, and could be activated most effectively by a reduced flavin adenine dinucleotide-regenerating system in an atmosphere of Hz or Nz. This was accomplished by the reduction of FAD with (a) reduced nicotinamide adenine dinucleotide and mammalian diaphorase or bacterial NADH dehydrogenase, or (b) platinum and Hz. Reduced riboflavin, FMNH2, and platinum plus Hz alone could replace FADH2. Dithionite activated the enzyme with a short lag period. Sulfhydryl compounds were needed for maximal effect in these activation procedures, but were not indispensable. Chorismate synthetase was activated slowly by NADH, or by thorough removal of oxygen, but only in the presence of thiols. The activation by NADH was probably due to trace amounts of flavins, since their removal abolished this effect. Preliminary evidence suggested that Fe++ may be the labile component of the enzyme responsible for the sensitivity to oxygen, although added Fe+-+ was also inhibitory.