CARBON DIOXIDE 



OXALOSUCCINIC CARBOXYLASE AND ISOCITRIC DEHYDROGENASE 



COOH COOH 



i I 



CO CHOH 



CH2 + CO2 + TPNH2 , HC— COOH + TPN (Ic) 



CH2 CH2 



I I 



COOH COOH 



a-Ketoglutaric acid /-Isocitric acid 



Since both reactions lb and Ic involve conversion of TPNH2 

 to TPN and vice versa, they can be followed spectrophotometrically in 

 either direction by allowing the reaction to take place in a quartz cell 

 and measuring the absorption of light at wave length 340 m^ by the 

 test solution. As mentioned above, reduced pyridine nucleotides 

 strongly absorb light of this wave length. The molar extinction coef- 

 ficient, which is defined by the equation: 



log hi I 

 a. — 



cl 



is 0.5644 X 10^ (cm. -/mole). For a transmittance of 95% and when 

 / = 1 cm., the concentration of reduced pyridine nucleotide would be 

 0.04 X 10"'' moles per cc. In the case of TPN with a molecular 

 weight of 743, 0.04 X 10"^ moles per cc. corresponds to 3 fxg. of TPN 

 per cc, or 0.8 Mg- of isocitric acid per cc. This indicates the great 

 sensitivity of the optical method and how suited it is for the study of 

 reactions of this type. 



By using this method, it has been possible to determine the 

 equilibrium constants of reactions la, lb, and Ic. The equilibrium 

 constant of reaction lb, A"b = (/-isocitrate) (TPN)/(oxalosuccinate) 

 (TPNH2), at/?H 7.0 and 22° C, is approximately 0.3. That of reac- 

 tion Ic, K\ = (/-isocitrate) (TPN) /(a-ketogIutarate)(C02)(TPNH2), 

 at the same pH and temperature is, on the average, 1.3 X lO"*. The 

 equilibrium constant of reaction la can be calculated from these two 

 values, since fi\ = (oxalosuccinate)/(a-ketoglutarate)(C02) = 

 Kc/Kx, = 0.5 X 10^^ Thus, tlie equilibrium of reaction la is so un- 

 favorable for carbon dioxide fixation that by this step alone only about 

 0.5% of the a-ketoglutarate would be carboxylated. 



