Functionally important residues of aromatic L-amino acid decarboxylase probed by sequence alignment and site-directed mutagenesis.
To identify functional residues of rat liver L-aromatic amino acid decarboxylase (AADC), we aligned the sequences of 13 group II amino acid decarboxylases and performed mutational analysis on the residues that were invariant or conservatively substituted. Replacements of His192, Asp252, Asp271, Ser296, Lys303, Tyr332, and Arg355 with alanine residues decreased the AADC activity (kcat/K(m)) by more than 10(4)-fold. Conservative replacements of [Asp252-->Glu], [Lys303-->Arg], and [Tyr332-->Phe] also resulted in decreases in activity by more than 10(4)-fold, indicating that both the chemical properties and the shape of these residues are essential for catalysis. The presence of a Schiff base between the amino group of Lys303 and the coenzyme pyridoxal 5'-phosphate is important for catalysis, probably at the transaldimination step. The enzyme activity was essentially unaffected by conservative mutation of [Arg355-->Lys], showing that the presence of a basic group at position 355 is necessary and sufficient for the catalysis. Replacement of [Thr246-->Ala], [His269-->Ala], and [Trp363-->Leu] yielded mutant enzymes that were 1-8% as active as the wild-type enzyme; these residues are not essential for the catalysis but are considered to contribute to the activity through conformational or other effects. The roles of the catalytically important residues of the group II amino acid decarboxylases probed in this study were discussed in the light of their relationship with the residues of other pyridoxal enzymes.
Ishii
S
S
Department of Biochemistry, Osaka Medical College.
Mizuguchi
H
H
Nishino
J
J
Hayashi
H
H
Kagamiyama
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011732:Pyridoxal Phosphate / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012545:Schiff Bases; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D012545:Schiff Bases; D011732:Pyridoxal Phosphate; D001142:Aromatic-L-Amino-Acid Decarboxylases