Acid-base chemistry of the reaction of aromatic L-amino acid decarboxylaseand dopa analyzed by transient and steady-state kinetics: Preferential binding of the substrate with its amino group unprotonated

Transient and steady-state kinetic analysis of the reaction of aromatic L-a mino acid decarboxylase (AADC), a pyridoxal 5'-phosphate- (PLP-) dependent enzyme, with its substrate dopa was carried out at various pH. The associat ion of AADC and dopa to form the Michaelis complex and the subsequent trans aldimination reaction to form the dopa-PLP Schiff base (external aldimine) were followed with a stopped-flow spectrophotometer. Combined with the stea dy-state k(cat) value, we could present a minimum mechanism for the reactio n of AADC and dopa. In the mechanism, the association of the aldimine-proto nated form of the enzyme (EH+) and the cr amino-group-unprotonated form of the substrate (S) is the main route leading to the Michaelis complex. In ad dition, 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 b e decreased in the Michaelis complex, promoting the conversion of EH+.SH+ t o 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 aminotransfer ase (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 pre valent species SH+ [Hayashi and Kagamiyama (1997) Biochemistry 36, 13558-13 569], 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 flexibil ity of the protein in the Michaelis complex, and a simulation analysis show ed that the presence of this route decreases the k(cat) value while increas ing the k(cat)/K-m value. It also suggested that AADC has evolved to suppre ss the minor route to the extent necessary to obtain the maximal k(cat) val ue at neutral pH.