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'-phosphate active site per subunit. In addition to catalyzing the decarboxylation of -aromatic amino acids, DDC also reacts with 5-hydroxytryptamine (5-HT), converting it to 5-hydroxyindolacetaldehyde 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 Kcat and Km values of this reaction were determined to be 0.48 min-1 and 0.47 mM, respectively. The NaBH4-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 Ki and Kinact 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.
Bertoldi
M
M
Istituto di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università di Verona, Strada Le Grazie, 37134 Verona, Italy.
Moore
P S
PS
Maras
B
B
Dominici
P
P
Voltattorni
C B
CB
D000447:Aldehydes / Q000378:metabolism; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D011994:Recombinant Proteins; D012701:Serotonin / Q000494:pharmacology*; D013057:Spectrum Analysis; D013552:Swine
D000447:Aldehydes; D065105:Aromatic Amino Acid Decarboxylase Inhibitors; D011994:Recombinant Proteins; D012701:Serotonin; D000641:Ammonia