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J. Biol. Chem., Vol. 277, Issue 39, 36357-36362, September 27, 2002

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Mutation of Tyrosine 332 to Phenylalanine Converts Dopa Decarboxylase into a Decarboxylation-dependent Oxidative Deaminase^*

Mariarita Bertoldi, Marco Gonsalvi, Roberto Contestabile, and Carla Borri Voltattorni^§

From the 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, and the  Dipartimento di Scienze Biochimiche "A. Rossi Fanelli" and Centro di Biologia Molecolare del Consiglio Nazionale delle Ricerche, Università "La Sapienza," 00185 Roma, Italy

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 CO₂ with concomitant O₂ 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 C 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.

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