Investigation of the nature of o-phthalaldehyde reaction with octopine dehydrogenase.

Abstract

The effect of o-phthalaldehyde on octopine dehydrogenase inactivation has been studied. o-Phthalaldehyde binds to the proximal cysteine and lysine residues of the enzyme leading to the formation of isoindole derivative. Double inhibition studies with o-phthalaldehyde and p-chloromercuricphenyl sulfonic acid have indicated that o-phthalaldehyde does not bind to the functional cysteine present at the active site. Protection experiments have shown that L-arginine prevented o-phthalaldehyde inactivation. This could be only due to the reaction of the amino group of L-arginine with o-phthalaldehyde as per the mechanism proposed elsewhere since L-arginine cannot bind to the enzyme prior to NADH. Other substrates such as pyruvate oR NADH could not prevent the o-phthalaldehyde reaction with the enzyme. Fluorescence spectral studies demonstrated that in the presence of externally added amino acid no isoindole derivative formation occurs. However, a characteristic isoindole derivative is formed in the presence of beta-mercaptoethanol although the enzyme does not lose its activity. This indicated that o-phthalaldehyde can bind with lysine of the enzyme and thiol of externally added beta-mercaptoethanol. Pyridoxal 5'-phosphate, a lysine specific reagent also binds to the enzyme giving the characteristic absorption and fluroescence peak at 325 nm and 395 nm respectively. However, no loss of enzyme activity was observed. On the basis of these experiments we would suggest that o-phthalaldehyde binds to non-essential cysteine and lysine residues present in close proximity which results in conformational changes leading to enzyme inactivation.