Rat liver aromatic L-amino acid decarboxylase: spectroscopic and kinetic analysis of the coenzyme and reaction intermediates.
The physiochemical properties of the coenzyme in rat liver aromatic L-amino acid decarboxylase (AADC) expressed in Escherichia coli have been studied by spectroscopic analysis of the enzyme, its reaction intermediates, and its complexes with substrate analogs. The enzyme, having one pyridoxal 5'-phosphate (PLP) per subunit, shows a prominent absorption maximum at 335 nm and a weaker one at 425 nm. The spectrum did not essentially change in the pH range of 6.0-8.0. When the coenzyme was excited at 335 nm, it emitted fluorescence primarily at 520 nm. The structure for the absorption at 335 nm was ascribed to the enolimine form of the PLP-lysine Schiff base. On the reaction of AADC with L-3,4-dihydroxyphenylalanine (L-dopa), the absorption of PLP showed biphasic changes before reaching a steady-state. Results of both pre-steady-state and steady-state kinetic analyses were consistent with the model that the reaction proceeds as shown in the equation: E + S<==>X1<==>X2-->E + P. The rate constant was determined for each step, and the Km value for L-dopa was obtained as 0.086 mM. The absorption spectra of the two intermediates, X1 and X2, were postulated from the calculation of the absorption changes during the first and the second steps of the reaction in which X1 and X2 showed an absorption maximum at 425 and 380 nm, respectively, with a concomitant decrease in absorbance at 335 nm. These predicted absorption spectra of X1 and X2 showed striking resemblances to those of AADC complexed with dihydroxyphenylacetic acid (DOPAc) and L-dopa methyl ester (DopaOMe), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Hayashi
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H
Department of Biochemistry, Osaka Medical College, Japan.
Mizuguchi
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Kagamiyama
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H
D000818:Animals; D001051:Apoenzymes / Q000302:isolation & purification; D001142:Aromatic-L-Amino-Acid Decarboxylases / Q000187:drug effects / Q000378:metabolism*; D001483:Base Sequence; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010659:Phenylhydrazines / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D001051:Apoenzymes; D010659:Phenylhydrazines; D011994:Recombinant Proteins; C030299:phenylhydrazine; D007980:Levodopa; D011732:Pyridoxal Phosphate; D001142:Aromatic-L-Amino-Acid Decarboxylases