Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5'-phosphate active site per subunit. In addition to catalyzing the decarboxylation of L-aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxy-indolacetaldehyde and ammonia. These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with high performance liquid chromatographic and mass spectroscopic analysis. The K-cat and K-m values of this reaction were determined to be 0.48 min-1 and 0.47 mM, respectively. The NaBH-4-reduced enzyme does not catalyze this reaction. Concurrent with this reaction, 5-HT inactivates DDC in both a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations, with K-i and K-inact values of 0.40 mM and 0.023 min-1, respectively. Protection from inactivation by 5-HT was observed in the presence of the active site-directed inhibitor 3,4-dihydroxy-D-phenylalanine. Inactivation with (2-14C)5-HT results in the incorporation of 1 mol of label/enzyme subunit. Taken together, these findings indicate that 5-HT is both a substrate and a mechanism-based inactivator with a partition ratio for product formation versus inactivation of 21. The absorbance, CD, and fluorometric features of 5-HT-inactivated DDC have also been characterized. A speculative mechanism for the reaction and inactivation consistent with the experimental findings is presented.
Mechanism-based inactivation of dopa decarboxylase by serotonin
Bertoldi M.;Moore P. S.;Dominici P.;Voltattorni C.
1996-01-01
Abstract
Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5'-phosphate active site per subunit. In addition to catalyzing the decarboxylation of L-aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxy-indolacetaldehyde and ammonia. These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with high performance liquid chromatographic and mass spectroscopic analysis. The K-cat and K-m values of this reaction were determined to be 0.48 min-1 and 0.47 mM, respectively. The NaBH-4-reduced enzyme does not catalyze this reaction. Concurrent with this reaction, 5-HT inactivates DDC in both a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations, with K-i and K-inact values of 0.40 mM and 0.023 min-1, respectively. Protection from inactivation by 5-HT was observed in the presence of the active site-directed inhibitor 3,4-dihydroxy-D-phenylalanine. Inactivation with (2-14C)5-HT results in the incorporation of 1 mol of label/enzyme subunit. Taken together, these findings indicate that 5-HT is both a substrate and a mechanism-based inactivator with a partition ratio for product formation versus inactivation of 21. The absorbance, CD, and fluorometric features of 5-HT-inactivated DDC have also been characterized. A speculative mechanism for the reaction and inactivation consistent with the experimental findings is presented.
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