CARBON DIOXIDE OR BICARBONATE AS SUBSTRATE OF NICOTINAMIDE-ADENINE DINUCLEOTIDE PHOSPHATE-LINKED ISOCITRATE DEHYDROGENASE AND 'MALIC' ENZYME By K. DALZIEL

1. A simple kinetic method was devised to show whether dissolved CO2 orHCO3ion is the substrate in enzyme-catalysed carboxylation reactions. 2. The timecourse of the reductive carboxylation of 2-oxoglutarate by NADPH, catalysed by isocitrate dehydrogenase, was studied by a sensitive fluorimetric method at pH 7-3 and pH 6-4, with large concentrations of substrate and coenzyme and small carbon dioxide concentrations. 3. Reaction was initiated by the addition of carbon dioxide in one ofthree forms: (i) as the dissolved gas in equilibrium with bicarbonate; (ii) as unbuffered bicarbonate solution; (iii) as the gas or as an unbuffered solution of the gas in water. Different progress curves were obtained in the three cases. 4. The results show that dissolved CO2 is the primary substrate of the enzyme, and that HCO3ion is at best a very poor substrate. The progress curves are in quantitative agreement with this conclusion and with the known rates of the reversible hydration of CO2 under the conditions of the experiments. The effects of carbonic anhydrase confirm the conclusions. 5. Similar experiments on the reductive carboxylation of pyruvate catalysed by the 'malic' enzyme show that dissolved CO2 is the primary substrate of this enzyme also. 6. The results are discussed in relation to the mechanisms of these enzymes, and the effects ofpH on the reactions. 7. The advantages of the method and its possible applications to other enzymes involved in carbon dioxide metabolism are discussed.