Mutation of tyrosine 332 to phenylalanine converts dopa decarboxylase into a decarboxylation-dependent oxidative deaminase.
A flexible loop (residues 328-339), presumably covering the active site upon substrate binding, has been revealed in 3,4-dihydroxyphenylalanine decarboxylase by means of kinetic and structural studies. The function of tyrosine 332 has been investigated by substituting it with phenylalanine. Y332F displays coenzyme content and spectroscopic features identical to those of the wild type. Unlike wild type, during reactions with l-aromatic amino acids under both aerobic and anaerobic conditions, Y332F does not catalyze the formation of aromatic amines. However, analysis of the products shows that in aerobiosis, l-aromatic amino acids are converted into the corresponding aromatic aldehydes, ammonia, and CO(2) with concomitant O(2) consumption. Therefore, substitution of Tyr-332 with phenylalanine results in the suppression of the original activity and in the generation of a decarboxylation-dependent oxidative deaminase activity. In anaerobiosis, Y332F catalyzes exclusively a decarboxylation-dependent transamination of l-aromatic amino acids. A role of Tyr-332 in the Calpha protonation step that catalyzes the formation of physiological products has been proposed. Furthermore, Y332F catalyzes oxidative deamination of aromatic amines and half-transamination of d-aromatic amino acids with k(cat) values comparable with those of the wild type. However, for all the mutant-catalyzed reactions, an increase in K(m) values is observed, suggesting that Y --> F replacement also affects substrate binding.
Dipartimento di Scienze Neurologiche e della Visione, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università degli Studi di Verona, Strada Le Grazie, 8, 37134 Verona, Italy.
Mariarita
M
Bertoldi
Marco
M
Gonsalvi
Roberto
R
Contestabile
Carla Borri
CB
Voltattorni
D000588:Amines / Q000737:chemistry; D000596:Amino Acids / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002384:Catalysis; D004296:Dopa Decarboxylase / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010101:Oxygen Consumption; D010649:Phenylalanine / Q000737:chemistry*; D011485:Protein Binding; D014443:Tyrosine / Q000737:chemistry*
D000588:Amines; D000596:Amino Acids; D014443:Tyrosine; D010649:Phenylalanine; D004296:Dopa Decarboxylase