VOL. 12 (1953) REVERSIBILITY OF GLUCOSE-6-PHOSPHATE OXIDATION 



101 



In the oxidation of glucose-6-phosphate to ribulose-5-phosphate and CO 2 with 

 purified Zwischenferment and phosphoghiconic dehydrogenase the limiting step appears 

 to be the hydrolysis of the lactone ring. In the presence of both dehydrogenases approxi- 

 mately 2 moles of TPN were reduced for each mole of glucose-6-phosphate present 

 (fig. I A). The first equivalent, however, was reduced much more rapidly than the second. 



0) 



C 



a. 



o 



V) 



c 1.0 



0) 



3 

 ^ 



c 

 .0 



t 



■4.8 



S -5.2 



S 

 o 



c 

 & 

 (J 



& 



-5.6 



-6.0 



10 

 Minutes 



5 

 Minutes 



Fig. I. The oxidation of glucose-6-phosphate by TPN in the presence oi Zwischenferment and phospho- 

 gluconic dehydrogenase. The reaction mixture (1.49 ml) contained 0.14 /<M of TPN, 0.031 fiM of 

 glucose-6-phosphate, 50 /iM of MgClj, 60 jxM of glycylglycine buffer, pH 7.4, and 0.063 mg of phos- 

 phoghiconic dehydrogenase, containing 0.23 units of Zwischenferment and 0.52 units of phospho- 

 gluconic dehydrogenase. TPN reduction was measured at 340 m// in a i.o cm cell. B represents the 

 logarithmic plot of the reaction from one-half minute to seven minutes. 



although the phosphogluconic dehydrogenase activity was more than twice the Zwischen- 

 ferment activity. With the amount of enzyme present in this experiment 6-phospho- 

 gluconate itself would be completely oxidized within the first minute; the relatively 

 slow reaction observed must represent the hydrolysis of phosphogluconolactone. The 

 second half of the reaction follows first order kinetics (fig. iB) from which it can be 

 estimated that the half time for hydrolysis of the lactone ring under these conditions 

 is about 1.5 minutes. 



DISCUSSION 



The experiments reported indicated that 6-phosphogluconaloctone (presumably on- 

 ly the S-lactone) can be enzymically reduced to glucose-6-phosphate, in the presence 

 of a source of reduced TPN. Together with evidence previously reported^ for the reduc- 

 tive carboxylation of ribulose-5-phosphate to 6-phosphogluconate these results provide 

 a mechanism for the direct fixation of CO 2 in hexose. The recent observations of 

 ViSHNiAC AND OcHOA^^ suggcst that photosynthetic mechanisms are able to provide the 

 reduced coenzyme, and thus the energy, for these reactions. The following reactions 

 would be involved in the formation of hexosemonophosphate by a reversal of its oxida- 

 tion: 



Ribulose-5-phosphate + COg + TPNH + H+ ^ 6-phosphogluconate + TPN (i) 



6-Phosphogluconate ^ 6-phosphogluconolactone -f H2O (2) 



6-Phosphogluconolactone -f TPNH -j- H+ ^ glucose-6-phosphate + TPN (3) 



With the enzyme preparations used in these experiments reaction^ appears to be the 



rate-limiting step. For this pathway for the oxidation of glucose-6-phosphate to have 



References p. 102. 



