April 5, 1954 



Cyclic Regeneration of Carbon Dioxide Acceptor 



1767 



However, if the glycolic acid is an indication of the 

 free two-carbon fragment, then the observation 

 that its increase in concentration (following reduc- 

 tion in COj pressure) is not as rapid as the increase 

 in RDP concentration suggests that the Cj com- 

 fjound is not as closely related to the carboxylation 

 reaction as the RDP. 



2. Origin of Ribulose Diphosphate. — If one 

 considers the principal labeling at short times of 

 PGA,^ RDP, SMP and the two hexose monophos- 

 phates^ as, respectively 



CH20© 

 CHOH 

 •**COOH 



PGA 



CHjOH 



Lo 



•CHOH 



I 

 ♦CHOH 



I 

 CHjO© 'CHOH 



CHOH 



CH,0<g) 

 RDP SMP 



•CHjO© 



I 



*c=o 



I 



"CHOH 



I 

 CHOH 



I 



C 



4 



c 



I 



c 



HMP 



it apj>ears that the ribulose is not derived entirely 

 from a Ce ^- Ci -f- C6 split or a C7 — >• C2 + Ce split. 

 No five carbon fragment of the hexose or the hep- 

 tose molecules contains the same distribution of 

 radiocarbon as ribulose. The combination of C3 

 with a labeled C2 fragment could account for the 

 observed radioactivity. However, some mecha- 

 nism for the labeling of the C2 fragment would be re- 

 quired. One such mechanism would be the break- 

 down of hexose simultaneously into three Cj frag- 

 ments,^' and since carbon atoms 3 and 4 of hex- 

 ose are labeled, a labeled Cj fragment might thus 

 be obtained. To our knowledge there exists no 

 precedent as yet for this type of reaction. 



Another way of accounting for the observed dis- 

 tribution of radioactivity which seems quite plaus- 

 ible in view of the rapidly accumulating enzymatic 

 evidence for the reverse reaction '''•^'"-■' is the forma- 

 tion of ribulose from sedoheptulose and triose. This 

 reaction could result in the observed labeling 



CH,OH •♦CHO 



I I 



=0 + CHOH 



I 



I 



*CHOH CH2O© 



I 

 •CHOH 



I 

 •CHOH 



I 

 CHOH 



CHjO© phospho- 

 SMP glyceraldehyde 



CHjOH 



I 



c— o -t- 



I 



•CHOH 



I 

 CHOH 



I 

 CHzO© 



ribulose 



•CHO 



I 

 •CHOH 



I 

 •CHOH 



CHOH 



I 

 CH2O© J 



ribose 



•C 



I 

 •C 



I 



•••c 



I 



c 



I 



c 



monophos- monophos- 

 phate phate 



If the ribose-5-phosphate and ribulose-5-phosphate 

 are then converted to RDP the resulting distribu- 



(21) H. GaffroD, E. W. Fager and J. L. Rosenberg, "Carbon Dioxide 

 Fixation and Photosynthesis," Symposia of the Society for Experi- 

 mental Biology (Great Britain), Vol. V, Cambridge University Press, 

 19S1. 



(22) B. Aadrod, R. S. Baudurslii, C. M. Greiner and R. Jang. J. 

 Biol. Chem.. SOI, 619 (1953). 



(23) B. L. Horecker and P. Z. Smymiotis, This Journal, T4, 212S 

 (1952). 



(24) B L Horecker and P Z. Smymiotis, itruf , It, 1009 (1963). 



tion of label would be that observed (carbon skele- 

 ton at right of reaction). 



3. Origin of Sedoheptulose.— The degradation 

 data appear to eliminate the possibility of formation 

 of sedoheptulose by a simple 6 + 1 or 6 -f 2 addi- 

 tion, if we assume that no special reservoirs of pen- 

 tose and hexose exist with distributions of radioac- 

 tivity different from those measured. A reverse 

 of the reactions proposed above for formation of 

 RDP would require segregation of ribose and ribu- 

 lose distributions as well as some other mechanism 

 for labeling the ribose in the manner shown. It 

 does seem likely that all the reactions involving 

 rearrangements of sugars and perhaps those in- 

 volving reduction of PGA as well are at least par- 

 tially reversible in the time of these experiments. 

 If all these compounds are intermediates in a cycle 

 of carbon reduction, then during steady state pho- 

 tosynthesis there will be a net "flow" of radiocarbon 

 in the "forward" direction, but the possibility that 

 the distribution of radiocarbon in later intermedi- 

 ates may reflect to some extent that of earlier inter- 

 mediates cannot be entirely ignored. 



The condensation of a triose with a C4 fragment 

 would give the observed distribution if the C4 frag- 

 ment is labeled in the carbon atoms 1 and 2 



CHjO© 



c=o 



♦CHji 



OH 



•CHO 



I 

 •CHOH 



I 

 CHOH 



I 

 CHsO© 



CHsO© 

 C= 



•i: 



-o 



DHAP 



HOH 



1 

 •CHOH 



I 

 •CHOH 



I 

 CHOH 



I 

 CH,0© 



Enzymatic evidence for this reaction and its re- 

 verse has been reported. ^''^^ 



4. Origin of the Four-Carbon Fragm«it. — Two 

 possible modes of formation of the four-carbon 

 fragment with the above labeling are a Cj + C3 

 addition, and a Ce -+ [C2] -|- [C4] split. The C, + 

 C3 addition which leads to malic acid produces a 

 C4 fragment labeled in the two terminal positions." 

 Therefore, the reduction of the dicarboxylic acid 

 formed as a precursor to malic acid could not result 

 in a C4 fragment with the C'^ distribution required 

 for the formation of 3,4,.5-C'* labeled sedoheptulose. 

 The rapid introduction of radiocarbon into malic 

 acid in earlier experiments* can be accounted for if 

 it is assumed that the reservoir size of malic acid, 

 depleted during the air flushing prior to the addi- 

 tion of HC'HDa", was increasing after the addition 

 of radiocarbon due to the increase in total CO2 

 pressure. Also, after the carboxyl group of PGA 

 and phosphoenolpyruvic acid have become appre- 

 ciably labeled, the mahc acid is doubly labeled. 



It is interesting to note that in the long term 

 "steady state" experiments in which the light was 

 turned off,' the mahc acid concentration dropped 

 when the light was turned off rather than increas- 

 ing as PGA concentration increased. If maUc acid 

 were an indicator of a four-carbon intermediate in 

 carbon reduction, the product of a second carboxyl- 



(25) B L. Horecker and P Z Smymiotis, ifriJ, 76, 2021 (1853). 



99 



