MECHANISM-BASED INACTIVATION OF DOPA DECARBOXYLASE BY SEROTONIN

Pig kidney dopa decarboxylase (DDC) expressed in Escherichia coli is a homodimeric enzyme containing one catalytically active pyridoxal 5'-p hosphate active site per subunit. In addition to catalyzing the decarb oxylation of L-aromatic amino acids, DDC also reacts with 5-hydroxytry ptamine (5-HT), converting it to 5-hydroxyindolacetaldehyde and ammoni a. These products have been identified by means of the enzymes alcohol dehydrogenase and glutamate dehydrogenase, together with high perform ance liquid chromatographic and mass spectroscopic analysis. The K-cat and K-m values of this reaction were determined to be 0.48 min(-1) an d 0.47 mM, respectively. The NaBH4-reduced enzyme does not catalyze th is reaction. Concurrent with this reaction, 5-HT inactivates DDC in bo th a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations, with K-i and K-ina ct 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-d irected inhibitor 3,4-dihydroxy-D-phenylalanine. inactivation with [2- C-14]5-HT results in the incorporation of 1 mol of label/enzyme subuni t. Taken together, these findings indicate that 5-HT is both a substra te and a mechanism-based inactivator with a partition ratio for produc t formation versus inactivation of 21. The absorbance, CD, and fluorom etric features of 5-HT-inactivated DDC have also been characterized. A speculative mechanism for the reaction and inactivation consistent wi th the experimental findings is presented.