title: Acid-base chemistry of the reaction of aromatic L-amino acid decarboxylase and dopa analyzed by transient and steady-state kinetics: preferential binding of the substrate with its amino group unprotonated.
abstract: Transient and steady-state kinetic analysis of the reaction of aromatic L-amino acid decarboxylase (AADC), a pyridoxal 5'-phosphate- (PLP-) dependent enzyme, with its substrate dopa was carried out at various pH. The association of AADC and dopa to form the Michaelis complex and the subsequent transaldimination reaction to form the dopa-PLP Schiff base (external aldimine) were followed with a stopped-flow spectrophotometer. Combined with the steady-state k(cat) value, we could present a minimum mechanism for the reaction of AADC and dopa. In the mechanism, the association of the aldimine-protonated form of the enzyme (EH(+)) and the alpha-amino-group-unprotonated form of the substrate (S) is the main route leading to the Michaelis complex. In addition, the association of EH(+) and the alpha-amino-group-protonated form of the substrate (SH(+)) to form a Michaelis complex EH(+).SH(+) was also found as a minor route. The pK(a) of the alpha-amino group of dopa was expected to be decreased in the Michaelis complex, promoting the conversion of EH(+).SH(+) to EH(+).S, the species that directly undergoes transaldimination to form the external aldimine complex. The association of EH(+) and S had been identified as a minor route for the reaction of aspartate and aspartate aminotransferase (AspAT), which has an unusually low pK(a) value of the aldimine and can use the aldimine-unprotonated form (E) of the enzyme for adsorbing the prevalent species SH(+) [Hayashi and Kagamiyama (1997) Biochemistry 36, 13558-13569]. The present study implies that, in most PLP enzymes that have a high pK(a) value of the aldimine like AADC, S preferentially binds to the enzyme (EH(+)). The minor route of EH(+) + SH(+) in AADC may be related to the flexibility of the protein in the Michaelis complex, and a simulation analysis showed that the presence of this route decreases the k(cat) value while increasing the k(cat)/K(m) value. It also suggested that AADC has evolved to suppress the minor route to the extent necessary to obtain the maximal k(cat) value at neutral pH.
authors.affiliation: Department of Biochemistry, Osaka Medical College, Takatsuki, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Hayashi
authors.forename: F
authors.initials: F
authors.lastname: Tsukiyama
authors.forename: S
authors.initials: S
authors.lastname: Ishii
authors.forename: H
authors.initials: H
authors.lastname: Mizuguchi
authors.forename: H
authors.initials: H
authors.lastname: Kagamiyama
mesh_terms: D000586:Amination; D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D004295:Dihydroxyphenylalanine / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007097:Imines / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008956:Models, Chemical; D011522:Protons; D051381:Rats; D015203:Reproducibility of Results; D013053:Spectrophotometry; D013379:Substrate Specificity
chemical_list: D007097:Imines; D011522:Protons; D004295:Dihydroxyphenylalanine; D001142:Aromatic-L-Amino-Acid Decarboxylases