266 Essays in Biochemistry 



since the reduction 1S of DHS to SA required an additional substrate, 

 isocitrate, and TPN for generating the required TPNH. 



Extracts of strain 83-24 of E. coli were able to convert hexose phos- 

 phates, ribose-5-phosphate, or sedoheptulose-7-phosphate (S-7-P) to 

 DHS in a yield of about 5%. 19 S-7-P plus FDP did not yield 

 significantly higher values. However, sedoheptulose-l,7-diphosphate 

 (SDP) 20 was almost quantitatively converted to DHS. 21 When SDP- 

 4,5,6,7-C 14 was incubated under these conditions, the SA obtained had 

 the same activity, and only in S-3, 4, 5, 6, showing that a four-carbon 

 fragment from SDP is utilized directly in shikimate formation. 21 



Table 1. Synthesis of DHS from E-4-P + PEP and from SDP * | 



Per Cent Conversion 



Substrates and Additions 

 E-4-P + 0.3 mM PEP 



+ fluoride + 0.3 fiM PEP 

 + fluoride + 0.5 nM 3-PGA 

 + iodoacetate + 0.3 nM PEP 

 + iodoacetate + 0.5 juM 3-PGA 



SDP 



+ fluoride 



+ fluoride + 0.5 /xM FDP 



+ fluoride + 0.5 mM 3-PGA 



+ fluoride + 0.5 nM pyruvate 



+ fluoride + 0.3 M M PEP 



+ iodoacetate 



+ iodoacetate + 0.5 juM FDP 



+ iodoacetate + 0.5 nM 3-PGA 



-f iodoacetate + 0.5 /xM pyruvate 



+ iodoacetate + 0.3 ^M PEP 



* Cell-free extracts were prepared by subjecting cells of freshly harvested 

 E. coli mutant 83-24 [B. D. Davis, J. Biol. Chem., 191, 315 (1951)] to sonic 

 vibration. The incubation mixtures contained 0.1 ml. of extract (2 mg. of 

 protein), 5 yM of MgCl 2 , 50 fiM of P0 4 s buffer pH 7.4, 0.25 nM of E-4-P + 

 0.3 nM of PEP, or 0.25 yM of SDP, + additions (10 mM of KF, or 0.5 mM of 

 iodoacetate) in a final volume of 1 ml. When iodoacetate was added, the solu- 

 tion, 0.95 ml., was preincubated at 37° for 15 minutes prior to the addition of 

 substrate. Following incubation at 37° for the indicated length of time aliquots 

 were removed for the bioassay of DHS with Aerobacter aerogenes mutant 

 A170-143S1 [B. D. Davis and U. Weiss, Arch. exp. Pathol, and Pharmakol, 220, 

 1 (1953)]. 



f Abbreviations: E-4-P, D-erythrose-4-phosphate; PEP, phosphoenolpyru- 

 vate; SDP, sedoheptulose-l,7-diphosphate; FDP, fructuse diphosphate; 3-PGA, 

 D-3-phosphogly eerie acid. 



