The role of biotin and sodium in the decarboxylation of oxaloacetate by the membrane-bound oxaloacetate decarboxylase from Klebsiella aerogenes.
The biotin-containing oxaloacetate decarboxylase from Klebsiella aerogenes catalyzed the Na+-dependent decarboxylation of oxaloacetate to pyruvate and bicarbonate (or CO2) but not the reversal of this reaction, not even in the presence of an oxaloacetate trapping system. The enzyme catalyzed an avidin-sensitive isotopic exchange between [1-14C]pyruvate and oxaloacetate, which indicated the intermediate formation of a carboxybiotin enzyme. Sodium ions were not required for this partial reaction, but promoted the second partial reaction, the decarboxylation of the carboxybiotin enzyme, thus accounting for the Na+ requirement of the overall reaction. Therefore, the 14CO2-enzyme which was formed upon incubation of the decarboxylase with [4-15C]oxaloacetate, could only be isolated if Na+ ions were excluded. Preincubation of the decarboxylase with avidin also prevented its labelling with 14CO2. The isolated 14CO2-labelled oxaloacetate decarboxylase revealed the following properties. It was slowly decarboxylated at neutral pH and rapidly upon acidification. The 14CO2 residues of the 14CO2-enzyme could be transferred to pyruvate yielding [4-14C]oxaloacetate. In the presence of Na+ this 14CO2 transfer was repressed by the simultaneous decarboxylation of the 14CO2-enzyme. However, Na+ alone was insufficient as a cofactor for the decarboxylation of the isolated 14CO2-enzyme, since this required pyruvate in addition to Na+. It is therefore concluded that the decarboxylation of oxaloacetate proceeds over a CO2-enzyme--pyruvate complex and that free CO2-enzyme is an abortive reaction intermediate. The activation energy of the enzymic decarboxylation of oxaloacetate changed with temperature and was about 113 kJ below 11 degrees C, 60 kJ between 11 degrees C and 31 degrees C and 36 kJ between 31--45 degrees C.
D001426:Bacterial Proteins / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D002262:Carboxy-Lyases / Q000378:metabolism*; D003653:Decarboxylation; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D010070:Oxalates / Q000378:metabolism; D010071:Oxaloacetates / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D013696:Temperature
D001426:Bacterial Proteins; D010070:Oxalates; D010071:Oxaloacetates; D001710:Biotin; D012964:Sodium; D002262:Carboxy-Lyases; C021915:oxalate decarboxylase