Multiple roles of the active site lysine of Dopa decarboxylase.
The pyridoxal 5'-phosphate dependent-enzyme Dopa decarboxylase, responsible for the irreversible conversion of l-Dopa to dopamine, is an attractive drug target. The contribution of the pyridoxal-Lys303 to the catalytic mechanisms of decarboxylation and oxidative deamination is analyzed. The K303A variant binds the coenzyme with a 100-fold decreased apparent equilibrium binding affinity with respect to the wild-type enzyme. Unlike the wild-type, K303A in the presence of l-Dopa displays a parallel progress course of formation of both dopamine and 3,4-dihydroxyphenylacetaldehyde (plus ammonia) with a burst followed by a linear phase. Moreover, the finding that the catalytic efficiencies of decarboxylation and of oxidative deamination display a decrease of 1500- and 17-fold, respectively, with respect to the wild-type, is indicative of a different impact of Lys303 mutation on these reactions. Kinetic analyses reveal that Lys303 is involved in external aldimine formation and hydrolysis as well as in product release which affects the rate-determining step of decarboxylation.
Bertoldi
Mariarita
M
Dipartimento di Scienze Morfologico-Biomediche, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università di Verona, 37134 Verona, Italy. mita.bertoldi@univr.it
Voltattorni
Carla Borri
CB
D015102:3,4-Dihydroxyphenylacetic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D000641:Ammonia / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003641:Deamination; D003653:Decarboxylation; D004296:Dopa Decarboxylase / Q000737:chemistry* / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism; D006860:Hydrogen Bonding; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D007980:Levodopa / Q000378:metabolism; D008239:Lysine / Q000378:metabolism*; D008956:Models, Chemical; D015394:Molecular Structure; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine
D015102:3,4-Dihydroxyphenylacetic Acid; D007980:Levodopa; C007430:3,4-dihydroxyphenylacetaldehyde; D011732:Pyridoxal Phosphate; D000641:Ammonia; D004296:Dopa Decarboxylase; D008239:Lysine; D000409:Alanine; D004298:Dopamine