It is important in considerations of the mechanism of COz fixation or decarboxylation to know whether or not the reactant is COn or HCO, (or H&02). Two methods ...
THE .JOURNAL
OF BIOLOGICAL
CHEMISTRY
Vol. 243. No. 14, Issue of July 25, PP. 3857-3863, 1968 Printed
The
in
U.S.A.
Carboxylation
of Phosphoenolpyruvate
ACTIVE SPECIES OF “COz” CARBOXYTRANSPHOSPHORYLASE,
I. THE
and
UTILIZED BY PHOSPHOENOLPYRUVATE AND PYRUVATE CARBOXYLASE*
Pyruvate CARBOXYKINASE,
(Received for publication, T. G. COOPER,~
T. T.
TCHEN,
HARLAND
G.
WOOD,
AND
March 4,
1968)
C. R. BENEDICT~
the Department of Chemistry, Wayne State University, Detroit, Michigan .$Sf?OZ?, and the Department Biochemistry, Case Western ReserveUniversity, Cleveland, Ohio 44106
From
SUMMARY
It is important in considerations of the mechanism of COz fixation or decarboxylation to know whether or not the reactant Two methods have been used t.o is COn or HCO, (or H&02). obtain such information. The first involves the use of carbonic anhydrase as exemplified by the studies of Krebs and Roughton (1). They presented evidence that COz is the product of decarboxylation of pyruvate, as catalyzed by pyruvate decarboxylase (EC 4.1.1.1). The evidence consisted of the * This study was assisted by Grants CA-08669, AM-05384, Atomic Energy Commission Contract AT-(30-l)-1320, and NSF Grant GB-2766. $ Present address, Department of Biological Sciences, Purdue University, Lafayette, Indiana 47907. $ Present address, Department of Plant Sciences, Texas A and M University, College Station, Texas 77843.
demonstration, by manometric methods, of an “overshoot” in COZ pressure during the decarboxylation, which was eliminated in the presence of carbonic anhydrase. This observation could be explained if CO2 is the product and if the rate of hydration of COZ is limiting. The CO2 under these conditions escapes into the gas phase, but as the substrate becomes limiting and the rate of the reaction decreases, the CO* is reabsorbed to attain the equilibrium shown below. CO* e co, (gaseous) (solution)
+ Hz0 * H&03 F? HCOs- + H+
(1)
In the presence of carbonic anhydrase the hydration of COz is so rapid that equilibrium is maintained throughout the decarboxylation. In contrast, if bicarbonate were the initial product, the evolution of CO* into the gas phase would be faster in the presence of carbonic anhydrase than in its absence, and there would be no overshoot of CO* pressure. Hans1 and Waygood (2), by use of this method and with a heavy suspension of disintegrated cells of Chlorella,or extracts from plants, presented evidence that CO2 is the primary product of decarboxylation of pyruvate, oxalacetate, glutamate, and cr-ketoglutarate. The second method of determining COZ species has involved the use of isO-bicarbonate. Kaziro ef al. (3) and Maruyama et al. (4) have presented evidence that HC03 (or HZC03) is the reactant in certain COZ fixations. They showed that al1 3 oxygens of HC1*03 were incorporated into the products, organic acids and phosphate, whereas if COZ were the reactant only 2 would be expected to be utilized. Kaziro et al. (3) used the biotin enzyme, propionyl-CoA carboxylase (EC 6.4.1.3), and found the equivalent of 1 I80 atom in the orthophosphate and 2 in the free carboxyl of the methylmalonyl-Cod. The results are illustrated in Reaction 2. CH3-CH*-CO-SCoA @SO-)-CO-SCoA
+ HCY03- + ATP i=! CHD-CH(C=+ Pi (containing 1 I80 atom) + ADP (containing
no excess IsO)
(2)
Maruyama et al. (4) used P-enolpyruvate carboxylase (EC 4.1.1.31) and observed that I80 was incorporated into the orthophosphate and oxalacetate in a ratio of 1:2, which is in accord with Reaction 3.
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Previous studies with propionyl coenzyme A carboxylase and with phosphoenolpyruvatecarboxylase using ‘*O-labeled bicarbonate have indicated that bicarbonate is the reactive speciesin these fixations of “C02.” We have investigated the speciesof CO:! in reactions catalyzed by pyruvate carboxylase, P-enolpyruvate carboxykinase, and P-enolpyruvate carboxytransphosphorylase. Since propionyl-CoA carboxylase and pyruvate carboxylase are biotin enzymes, they would be expected to have similar mechanisms. Likewise, the reactions catalyzed by P-enolpyruvate carboxykinase and carboxy transphosphorylase are, in some respects, similar to that of P-enolpyruvate carboxylase, and it has been suggestedthat bicarbonate might be the reactant in each case. By means of radiochemical and spectrophotometric techniques, we have obtained evidence that the active speciesin the carboxykinase and carboxytransphosphorylase reactions is CO2 and not bicarbonate. Bicarbonate appears to be the active species in the pyruvate carboxylase reaction, in conformity with the results obtained with propionyl-CoA carboxylase.
of
Carboxylation
3858 P-Enolpyruvate
of Phosphoenolpyruvate
+ HCY803- ---) 18-0180C-CH-CO-COO+ Pi (containing
an I80 atom)
(3)
Although the I80 results appear quite unequivocal, the method does involve one basic assumption. It is that OH- or water at the active site of the enzyme is in equilibrium with the solvent water. The reaction could occur as illustrated in Reaction 4 where E is the enzyme, P-Pyr is P-enolpyruvate, OA is oxalacetate, and P-OA is P-enoloxalacetate. OH
OH
/ -
E+OH--+E
co2
/
OH / E-CO2 \
+ P-Pyr
E-CO2
(4)
/ +E+OA
+Pi
\
P-Pyr
P-OA
P-Enolpyruvate
+ CO2 + GDP
1
Mn2f (5) oxalacetate
P-Enolpyruvate
+ CO2 + Pi
