RGD Reference Report - 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. - Rat Genome Database

RGD Reference Report - 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. - Rat Genome Database

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General

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.
Authors:	Hayashi, H Tsukiyama, F Ishii, S Mizuguchi, H Kagamiyama, H
Citation:	Hayashi H, etal., Biochemistry. 1999 Nov 23;38(47):15615-22.
RGD ID:	4139897
Pubmed:	PMID:10569946 (View Abstract at PubMed)
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.

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Molecular Function

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Term	Qualifier	Evidence	With	Reference	Notes	Source	Original Reference(s)
amino acid binding		IDA		4139897	amino acid derivative and L-DOPA	RGD
aromatic-L-amino-acid decarboxylase activity		IDA		4139897		RGD

Objects Annotated

Genes (Rattus norvegicus)

Ddc (dopa decarboxylase)

Additional Information