PACIFIC SOUTHWEST ASSOCIATION OF CHEMISTRY TEACHERS PHYTOSYNTHESIS: FIRST REACTIONS' ANDREW A. BENSON University of California, Berkeley, California
T H E reactions of phytosynthesis include all metabolic reactions in the plant which result in the synthesis of organic compounds. Of primary importance among these are the reactions of photosynthesis which store the energy of light as chemical energy by converting carbon dioxide and water through a series of intermediate compounds to carbohydrate and oxygen. Thus photosynthesis supplies both the materials and the energy for all other reactions of phytosynthesis. The studies of photosynFOOH 49 thetic C1402fixation in this :CHOH 25 laboratory have now led to &OH 26 an understanding of the HEXOSE first steps of carbohydrate c,,~, 52 synthesis. From these few G.C, 25 D compounds are derived the C, ,C, 24 multitude of compounds in the plant. Thefundamental procl5 SEC' BARLEY ess of photosynthesis is F~F.. 1. c ~ ~ d ~ l i in b ~the t iconversion ~ ~ of electro-
--
cates that two C3 molecules condense, head to head, to give a Cs. As time progresses, the a and B carbons of PGA acquire CI4 and the 1,2- and 5,6-carbon atoms of the hexoses become labeled. The typical distribution of label is given in Figure 1. Such data are obtained by chemical degradation of pure radioactive compounds separated from each other by two-dimensional paper chromatography. I t is now clear that the plant has made sucrose by a sequence of reactions similar if not identical to the process of glycolytic breakdown of sugars in animal tissue and yeast. In fact, each of the intermediates has been identified and observed to acquire radioactivity in the sequence shown below.
b,"*"
P-glycerie acid
"'
energy of the reducing agent(s) required for carbon dioxide reduction. A plausible proposal for the mechanism of this puzzling process has been advanced from our laboratory2 and is supported by a growing collection of circumstantial, if not positive, evidence. It is the function of thisphotochemically produced reducing power in phytosynthesis to which this survey is addressed. The major first product of CI4O2fixation by all plants is carhoxyl-labeled phosphoglycerate. Phosphoglyceric acid (PGA) becomes the major radioactive product. The first hexoses formed are 3,4-labeled. This indi'Based on a paper given at the meeting of the Southern Section of the Pacific Southwest Association of Chemistry Teachers, December 5, 1953, Immaculate Heart College, Los Angeles. The work deserihed in this paper was sponsored by the U. S. Atomic Energy Commission. This paper is a review of recent work in this laboratory, most of which is described in more detail elsewhere; BASSHAM, J. A,, A. A. BENSON,L. D . KAY,A. 2. HARRIB,A. T. WILSON,AND M. CALVIN,J. Am. Chem. Soe., 76, 1760 (1954). 'CALVIN,M.,Chem. Eng. News, 31, 1622, 1735 (1953). 484
C&OQ
H t o P-glyceraldehyde
H&OH &=O
HAoH dihydroxyacetone-P
-
H o cI H ~
4
0
~
6H20Q fructose1,6-dip CHO
HbOH
-
CbOB H4OH
Starch
4
sucrose phosphate
1
sucrose
As radiocarbon saturates successive reservoirs of
SEPTEMBER, 1954
485
these intermediates i t would be expected that specific radioactivity (concentration of CL4)would be greater for fructose phosphates than for glucose phosphates. This is verified experimentally by an examination of the earliest labeled sucrose. Upon acid hydrolysis and chromatography of the two hexoses, the fructose moiety contained the great.er part of the C14 (Figure 2). The exact mechanism of the enzymatic synthesis of sucrose is being studied in several laboratories and soon may be well understood. The carboxylation reaction by which phosphoglycerate is formed from CO1 and some constantly generated acceptor molecule has occupied our attention for some time. The biochemistry of microbiological or animal metabolism offered few clues to the nature of the cyclic process for making the carboxylation substrate. Such clues appeared in radiograms of short photosynthesis in c1402. The intermediates of sucrose synthesis were not the only phosphorylated sugars to acquire label in the first seconds of photosynthesis in C'40~. Sedoheptulose, previously found only in succulent plants such as sednms, and ribulose, known only as an uncrystallized synthetic sugar, became labeled in all photosynthetic organisms. They had not been observable before the advent of the tracer method with its great sensitivity for detecting their low concentrations (lo-' M ) and the paper chromatographic method for separating efficiently the numerous sugars and their phosphate esters. The stereostructure of sedoheptulose and ribulose bears little relationship to that of the hexoses. By splitting a glycolaldehyde fragment (Cz) from sedoheptulose, however, one obtains ribose, the epimer of ribulose. Further, ribulose might lose a CZto give triose, which we see is the reduction product of PGA. These relationships led t o the disc&ery of a ubiquitous enzqme system, transketolase, in laboratories of B. L. ~ o r e i k e r 3 and E. Racker. Their discovery of the transketolase enzyme supported and clarified our proposal of its function in photosynthesis. with transketolase, the acyloin-type cleavage of ketoses allows a transfer of glycolyl groups from one phosphorylated aldose to another.
phosphate
phosphate
These phosphorylated sugars have been isolated from the products of brief CL40zh a t i o n experimenh and chemically degraded to determine the C1' distribution 'HOREC~ER, B. L., P. Z. SMYRNIOTIS, AND H. KLENOW, J. Bid. Chem., 205, 661 (1953).
Figure 2.
Hydrolysate of Sucrose Formed During 15 Seconds Photosynthesis in C"On by Barley Seedling Leaves
within each molecule. The results for products offthe Erst few seconds of photosynthesis are schematically summarized :
,l Ir
triose
hexose
sedoheptulose
rihulose
I n time, of course, they all become uniformly CI4labeled. The observed labeling of the C5 molecule does not appear to result from Cz cleavage from CT. Transketolase, however, catalyzes CZ transfer from C, t o a C3aldehyde to give:
H&OH
BC'OH
o*=o
A-0
&OH
+
H&OH
(
H9CaOH
-- H ~ * * O H
JOURNAL OF CHEMICAL EDUCATION %
h *'>T*
“zO
N)P PPWI
td
co2
* 1~10% ~ D S R U T E
B4
A S a a r Rearrwement.
r i p r e 3.
I
cyclic pathway forR w n e m t i i n of Carbon Diodde Accaptor of ~ h ~ t ~ ~ ~ ~ t h ~ i s
This accounts for the pentose laheling, but we must have a mechanism for the heptose labeling. Its trans Of and suggested that it was formed, like fructose, by aldol condensation of eryth( ~ 4 ) with glyceraldehyde-3-phosro~e-4-~hos~hate phate (C3): H&OQ
H,COQ
L o
L o H,&*oH
+
-
Hoh*H HA*oH
Hca=o
HboH
HLoH
HcoH
HAoH
H , OQ
I
A
H1 OQ
The obvious source of such a 1,2-labeled erythrose phosphate among the first products of phytosynthesis is fructose-6-phosphate. CHO HboH
CH20H
+
4=0
HOOK Ha OQ
-
transketolase -
CHO* HboH*
CHSOH
+
&o
minutes, whereupon the reservoirs of the early photosynthetic intermediates became saturated with radiocarbon and the radioactivity in each compound became a measure of its concentration. In experiments by Mr. A. T. Wilson of this laboratory, the COz pressure was suddenly dropped from 1 per cent to 0.003 per cent. Small samples of algae were withdrawn from the illumination vessel every five seconds and their components separated on paper chromatograms. The radioactive arias, d e h e d by radiograms, were counted with a IargeGeiger tube. The concentration of phosphoglycerate and ribulose diphosphate showed profound changes. The concentration of phosphoglycerate, the carboxylation went down, as would he expected the carboxylation step (Figure 3) was blocked. Rihulose diuhos~hateconcentration. on the other hand, rose rapidly. When the COz pressure is kept constant and the light is turned off the cycle is blocked a t the reduction step and phosphoglycerate accumulates. Rihulose diphosphate and triose phosphate concentrations drop markedly. In fact, several oscillations in these concentrations were observed, as might he characteristic of any electrical or hydraulic closed circuit where an external condition is suddenly changed. Rihulose diphosphate is the largest Cs reservoir of most plants and it seems to represent the outlet for ribulose5-phosphate formed in the transketolase system. The phosphorylating power somehow developed during photosynthesis drives this synthesis: Ribulose-5-P
+ ATP
-
Ribulose-1,5-P
+ ADP
In considering ribulose-1,5-diphosphate as a possible C 0 2 acceptor we notice that it cannot assume a ketal form, as does fructose diphosphate, but must have a true carbonyl group. This might well enolize and add carbon dioxide to form an unstable or-hydroxy-P-keto acid. ~-
H ~ O Q HsCOQ
.oL
This hypothesis was elegantly proved by E. Racker, et 0 1 . . ~with crvstalline transketolase and m r e fructose- H ~ O H 6-phbsphate. "As soon as the plant has produced some labeled fructose-6-phosphate, then, there is a widespread equilibration of label among the ketoses and it becomes H ~ O B impossible to define the path of carbon in such an arbitrary system. The general trend of this synthetic system is towards the production of ribulose phosphate. Apparently this is the precursor of the C 0 2 acceptor and must he produced constantly. The glycolyl enzyme complex produced by each transketolase operation condenses with triose phosphate, one of the most available free alde- L hydes. unstable a-hydroxyWe are suggesting that such a pentose is required as 8-keto acid the C 0 2 acceptor of photosynthesis. Such a hypothesis is verified experimentally in the following manner. Hydration of the CrCs bond in such a keto acid should Green algae (Scenedesmus) were given C"Oa for twenty be possible and would lead to one molecule of labeled 4 RACRER, E., G . DE LA HABA, AND I.G. L E D E R , A ~ c ~ . B ~ ophosphoglycerate &~. and One One. time photosynthesis with C1402progresses the acceptor heand Biophys., 48,238 (1954).
:d",",$&
SEPTEMBER 1954
comes labeled and both phosphoglycerates contain 4 The chance in free energy of this reaction has been estimated to be -5 1 5 kg.-cal. and is extremely favorable compared to many other possible carboxylation reaations. This possibility of low-energy carboxylation arises because of oxidation of C3 from a -CHOH to a -COOH group. The cyclic system, then, is currently considered to be represented by Figure 4. While the existence of such a mechanism for the primary COXfixation step of photosynthesis is not yet certain: it would seem that when biochemical trausfor-
-
o2 IWSI ,
hr/ (quanturn~
w -
Figure 4.
chi*
pmposod Cych fo= Carbon Reduction in Photaynthesis
is now possible to reproduce the casboxylation step in a cell-free system (QUAYLE,J. R., R. C . FULL%A. A. BENSON, AND M. CALVIN, J. Am. Chem. Soc., 76, in press (1954)). Rad~oactive phosphoglycerate was formed from C ~ Q md ,ibu~08e diphosphate in the presence of 8 plant enzyme preparation. "It
roi
mations were being selected it should have merited the consideration of the plant world. It is particularly interesting that this reaction, through which almost all of the earth's reduced carbon passes, has not ~ ebeen t clearly defined.
