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J. Biol. Chem., Vol. 262, Issue 14, 6552-6563, May, 1987

CO2 adducts as reactive analogues of carboxylate substrates for aconitase and other enzymes of carbohydrate metabolism

DJ Porter, TA Alston and HJ Bright

The CO2 adducts resulting from N-, O-, and S-carboxylation of suitable precursors are close analogues of carboxylate substrates in which -NH- CO2-, -O-CO2-, or -S-CO2- replaces -CH2-CO2- in the physiological substrate, -NOH-CO-2 replaces -CHOH-CO-2 and O-CO2- replaces -O-PO3H- R- XH + CO2 in equilibrium with R-X-CO2- + H+ X(-XH = -NH2, -NHOH, -OH or - SH). We find that aconitase catalyzes the CO2-dependent dehydration of N-hydroxy-DL-aspartate and erythro-beta-hydroxyl-L-aspartate with respective kcat values 62 and 90% of kcat for citrate and Km values of 3.6 and 3.2 mM, respectively. The CO2 adducts (carbamates) of the precursors would be structural and stereo analogues of the physiological substrate isocitrate. Detailed kinetic analyses of the behavior of intermediates and products show that aconitase catalyzes the formation of the enzyme-bound CO2 adducts from enzyme-bound precursors and CO2 and directs them, as well as the preformed CO2 adducts, into alpha,beta water elimination reactions formally identical to the isocitrate/cis-aconitate reaction. Six other enzymes of carbohydrate metabolism (succinate thiokinase and isocitrate, glucose-6- phosphate, succinate semialdehyde, glutamate, and malate dehydrogenase) utilize CO2 adducts as reactive substrate analogues. At least one of these (glucose-6-phosphate dehydrogenase) catalyzes the formation of the enzyme-bound CO2 adduct (presumed to be D-glucose 6-carbonate in this case) from enzyme-bound precursor (D-glucose) and CO2 in the manner of aconitase. The case of malate dehydrogenase is unique because the reactive malate analogue, -O2C-O-CHOH-CO-2, arises from nucleophilic attack of HCO-3 on the carbonyl of glyoxylate, rather than electrophilic attack of CO2 on the hydrated carbonyl of glyoxylate.
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