1768 



Bassham, Benson, Kay, Harris, Wilson and Calvin 



Vol. 76 



ation, then one would expect its concentration to 

 increase in the dark for two reasons. First, there 

 no longer is reducing power which would reduce 

 the carboxylation product to sugar if this product 

 were an intermediate in CO2 reduction. Second, 

 the rate of formation of malic acid should increase 

 since this rate depends on the CO2 concentration 

 (which remains constant), and the concentration 

 of phosphoenolpyruvic acid (which increases paral- 

 leUng the PGA concentration). The decrease in 

 malic acid concentration could be easily explained 

 on the basis of the proposed light inhibition of py- 

 ruvic acid oxidation.' The cessation of illumina- 

 tion should permit increased pyruvic acid oxidation, 

 thus providing more acetyl-CoA, which can react 

 with oxaloacetic acid derived from malic acid. 



It is possible that there is a different "second 

 carboxylation" (Cj + Ci) leading eventually to a 

 four-carbon fragment which can react with those 

 to give sedoheptulose, but there seems to be no 

 evidence whatever for such a reaction at present. 

 Moreover, such a reaction should lead in short 

 times to a four-carbon fragment somewhat more 

 labeled in the terminal carbon position than in the 

 second carbon position due to dilution of the carbon 

 introduced in the first carboxylation reaction by 

 the PGA and triose reservoirs. This is not the 

 case — in fact in the very shortest times the ter- 

 minal carbon position of the hypothetical d frag- 

 ment (carbon four of sedoheptulose) is actually less 

 labeled than the second position, at least in the soy- 

 bean experiments. 



The most likely source of the C4 fragment seems 

 to be a Co -► [C*] -f [C2] split. Trioses could then 

 react with [C4] and [C2] to give sedoheptulose and 

 ribulose, respectively. One possible formulation of 

 these reactions would be 



C 



I 

 C= 



4 

 4 



•CHO 

 =0 -I- CHOH 

 CHsO© 



•C" 



I 

 •C 



CH.O© 



+ c=o — 



•CHjOH 



CH,OH 



c=o -f 



I 



•CHOH 



I 

 CHOH 



CH.O© 



CH,0© 



I 



c=o 



I 



•CHOH 



I 

 •CHOH 



:hoh 



HOH 



CHjO© 



The first reaction as written above would be a 

 transketolase reaction of the type reported by 

 Racker, et al.,^" who found that this enzyme splits 

 ribulose-.")-phosphate, leaving glyceraldehyde-.3- 

 phosphate and transferring the remaining two 

 carbon atoms to an acceptor aldehyde phosphate of 

 2-, 3- or 5-carbon atoms. No mention was made of 

 the effect of transketolase on ribulose-5-phosphate 

 with erythrosc-4-phosphate which would result in 



the formation of fructose phosphate by a reaction 

 which is just the reverse of the Ce-*- [C2] + [Ci] split 

 written above. ^' 



The labeling of carbon number 4 in sedoheptulose 

 observed in the case of the very short periods of 

 photosynthesis with soybean leaves seems to cast 

 some doubt on the Cj -»■ [C2] -f [C4] split unless 

 one can assume that the Ce which splits is itself 

 not symmetrically labeled at the shortest times, due 

 to different specific activities of the two trioses 

 which react to give hexose 



CH,0© 



CHOH 



I 

 •**COOH 

 PGA 



CHjO© 



2[H 



CHjO© 



, I 

 >CHOH 



- incomplete - 

 -equilibration- 



I 



•*CHO 



It 



later, hence 

 more complete 

 equilibration 



w 



CHsO© 



CHjO© 



. I 

 C=0 



" I 



•CHjOH 



F-l,6-DiP 



\ 



CHjOH 



I 



c=o 



CH,OH 



I 



c=o 



•CHOH 



I 

 ••CHOH 



I 

 CHOH 



CHjO© 



••CHO 



I 

 CHOH 



I 



I 

 •CHOH 



I 



CHOH 



CHjO© I 



CH2O'" 



c=o C=0 



••CHOH "CHjOH 



•CHOH 



I 

 ••CHOH < 



CHOH 



CH,0© 



Degradation of fructose from the 0.4- and 0.3- 

 sec. experiments showed no significant difference 

 between the two halves of fructose. It is quite 

 possible, however, that the differences in denatura- 

 tion rates of various enzymes mentioned earlier 

 may influence the results in these short times. 



Combining these reactions with others aheady 

 proposed we have the following cyclic path of car- 

 bon reduction during photosynthesis. The car- 

 bon fragments specified only by the number of car- 

 bon atoms in their chains are all at the sugar level 

 of reduction 



3Ci -I- 3C0j — 



121H 

 6PGA 



2C, - 



C, -I- 2C, - 



C, + C, 



6PGA 



> 6C, 



C, 



Cs-h C, 

 ■>2C. 



The net reaction for each turn of the cycle is 



12 (HI -f 3C0,- 



C.H,0, -I- 3H,0 



The operation of this cycle is illustrated in Fig. 7. 



5. Energetics of the Carbon Reduction Cycle. — 

 That the enzymatic rearrangements of sugars re- 

 quires no additional supply of energy in the form 

 of ATP or other sources seems to be indicated by 

 the experiments with isolated and partially purified 

 enzyme preparations in which such rearrangements 

 have been carried out without the addition of 

 energy donors. The free energy change of the car- 

 boxylation reaction can be roughly estimated. Es- 

 timating the free energy difference between ribose- 



(26) Since this was written, a private communiration from Dr. 

 Racker has informed us that he has observed this reaction with F-6-P 



100 



