REACTION OF DOPA DECARBOXYLASE WITH ALPHA-METHYLDOPA LEADS TO AN OXIDATIVE DEAMINATION PRODUCING 3,4-DIHYDROXYPHENYLACETONE - AN ACTIVE-SITE-DIRECTED AFFINITY LABEL

Dopa decarboxylase (DDC) catalyzes the cleavage of alpha-methylDopa in to 3,4-dihydroxyphenylacetone and ammonia, via the intermediate alpha- methyldopamine, which does not accumulate during catalysis, The ketone has been identified by high-performance liquid chromatography and mas s spectroscopic analysis, and ammonia by means of glutamate dehydrogen ase. Molecular oxygen is consumed during the reaction in a 1:2 molar r atio with respect to the products. The k(cat) and K-m of this reaction were determined to be 5.68 min(-1) and 45 mu M, respectively. When th e reaction is carried out under anaerobic conditions, alpha-methyldopa mine is formed in a time-dependent manner and neither ammonia nor keto ne is produced to a significant extent. The reaction is accompanied by a time- and concentration-dependent inactivation of the enzyme with k (inact) of 0.012 min(-1) and K-i of 39.3 mu M. Free 3,4-dihydroxypheny lacetone binds to the active site of DDC and inactivates the enzyme in a time- and concentration-dependent manner with a k(inact)/K-i value similar to that of alpha-methylDopa. D-Dopa, a competitive inhibitor o f DDC, protects the enzyme against inactivation. Taken together, these findings indicate the active site directed nature of the interaction of DDC with 3,4-dihydroxyphenylacetone and provide evidence that the k etone generated by the reaction of DDC with alpha-methylDopa dissociat es from the active site before it inactivates the enzyme. Inactivation of the enzyme by ketone followed by (NaBH4)-H-3 reduction and chymotr yptic digestion revealed that the lysine residue which binds pyridoxal 5'-phosphate (PLP) in the native enzyme is the site of covalent modif ication. Together with the characterization of the adduct released fro m the inactivated DDC, these data suggest that the enzyme is inactivat ed by trapping the coenzyme in a ternary adduct with ketone and the ac tive site lysine. As recently reported for serotonin (5-MT) [Bertoldi, M., Moore, P. S., Maras, B., Dominici, P., and Borri Voltattorni, C. (1996) J. Biol. Chem, 271, 23954-23959], the conversion of dopamine (D A) into 3,4-dihydroxyphenylacetaldehyde and ammonia catalyzed by DDC i s accompanied by irreversible loss of decarboxylase activity. However, the comparison between the absorbance, fluorescence, and CD features of DDC after 5-HT- or 3,4-dihydroxyphenylacetone-induced inactivation shows that a different covalent adduct is formed between either of the se two molecules and DDC-bound PLP.