title: An anomalous side reaction of the Lys303 mutant aromatic L-amino acid decarboxylase unravels the role of the residue in catalysis.
abstract: Aromatic L-amino acid decarboxylase (AADC) in which the pyridoxal 5'-phosphate (PLP)-binding residue Lys303 was replaced by an alanine residue is virtually inactive as a catalyst. On reaction of the normal substrate L-dopa with this mutant AADC, the absorption at around 330 nm gradually increased with concomitant decrease of the absorption of the free PLP molecule at 390 nm. Analysis of the 330-nm absorbing species on HPLC and spectrophotometry showed that it is a 1:1 adduct of PLP and dopamine, probably the Pictet-Spengler type adduct formed from the PLP-dopamine Schiff base. The product dopamine was not found outside of the enzyme, showing that the PLP-dopamine Schiff base undergoes adduct formation rather than yields product dopamine. The Pictet-Spengler adduct of PLP with L-dopa was not detected, whereas the corresponding adduct was formed when the carboxylate group of L-dopa was esterified with a methyl group to block the decarboxylation reaction. This suggests that the PLP-L-dopa Schiff base may undergo the Pictet-Spengler reaction, but its rate is much smaller than that of decarboxylation. These results indicate that Lys303 is not essential for the decarboxylation step, but has an important role in the product release, and possibly the transaldimination process.
authors.affiliation: Department of Biochemistry, Osaka Medical College, Takatsuki.
authors.forename: J
authors.initials: J
authors.lastname: Nishino
authors.forename: H
authors.initials: H
authors.lastname: Hayashi
authors.forename: S
authors.initials: S
authors.lastname: Ishii
authors.forename: H
authors.initials: H
authors.lastname: Kagamiyama
mesh_terms: D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D016296:Mutagenesis; D051381:Rats
chemical_list: D001142:Aromatic-L-Amino-Acid Decarboxylases; D008239:Lysine