Interactions between carbonic anhydrase and some decarboxylating enzymes as studied by a new bioelectrochemical approach.
This work presents the results of a study, carried out by recently developed amperometric bioelectrodes, on the interactions between carbonic anhydrase (CA) and the decarboxylating enzymes arginine decarboxylase (ADC), L-lysine decarboxylase (LDC), and L-ornithine decarboxylase (ODC). These are all pyridoxal-phosphate dependent enzymes and catalyze the decarboxylation reaction of the respective amino acids, to give carbon dioxide and the corresponding diamine (agmatine, cadaverine, and putrescine, respectively). The rate of each decarboxylase catalyzed reaction was measured by monitoring the production of the respective diamine by a plant tissue diamino oxidase (DAO) based bioelectrode. DAO is the enzyme which catalyzes the oxidation of agmatine, cadaverine, and putrescine with the production of NH and H2O2. DAO-based bioelectrodes consist of an amperometric H2O2 electrode, coupled to the biocatalytic membrane formed by a whole plant tissue (lentil cotyledon) containing the enzyme DAO, immobilized on a dialysis membrane by polyazetidine prepolymer (PAP). The bioelectrodes were calibrated and characterized in standard solutions of agmatine, cadaverine, and putrescine. Kinetic studies to measure decarboxylase activity were performed in the presence of different concentrations of ADC, LDC, and ODC, resulting in a lowest detection limit of 10, 25, and 10 U l(-1), respectively. The effect of bovine CA II (bCAII) was evaluated in the presence of 500 U l(-1) of each decarboxylase, showing a marked increase of the rate of the decarboxylation reaction. These results suggest that (i) CA can be used to enhance the performance of decarboxylase-based biosensors, and (ii) it possibly plays further physiological roles, acting synergistically, at specific cellular and subcellular sites, with low-activity decarboxylating enzymes.
Botrè
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Dipartimento di Gestione e Controllo delle Merci e del loro Impatto sull'Ambiente, Università La Sapienza, Rome, Italy. botre@.uniroma1.it
Mazzei
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D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry*; D002262:Carboxy-Lyases / Q000737:chemistry*; D002417:Cattle; D004563:Electrochemistry / Q000379:methods*; D004566:Electrodes; D004912:Erythrocytes / Q000201:enzymology; D007700:Kinetics; D009955:Ornithine Decarboxylase / Q000737:chemistry*; D010944:Plants / Q000201:enzymology
D002262:Carboxy-Lyases; D009955:Ornithine Decarboxylase; C021520:lysine decarboxylase; C019722:arginine decarboxylase; D002256:Carbonic Anhydrases