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Structures of Apo- and Holo-Tyrosine Phenol-lyase Reveal a Catalytically Critical Closed Conformation and Suggest a Mechanism for Activation by K+ Ions (CROSBI ID 124789)

Prilog u časopisu | izvorni znanstveni rad | međunarodna recenzija

Milić, Dalibor ; Matković-Čalogović, Dubravka ; Demidkina, Tatyana V. ; Kulikova, Vitalia V. ; Sinitzina, Nina I. ; Antson, Alfred A. Structures of Apo- and Holo-Tyrosine Phenol-lyase Reveal a Catalytically Critical Closed Conformation and Suggest a Mechanism for Activation by K+ Ions // Biochemistry (Easton), 45 (2006), 24; 7544-7552. doi: 10.1021/bi0601858

Podaci o odgovornosti

Milić, Dalibor ; Matković-Čalogović, Dubravka ; Demidkina, Tatyana V. ; Kulikova, Vitalia V. ; Sinitzina, Nina I. ; Antson, Alfred A.

engleski

Structures of Apo- and Holo-Tyrosine Phenol-lyase Reveal a Catalytically Critical Closed Conformation and Suggest a Mechanism for Activation by K+ Ions

Tyrosine phenol-lyase, a tetrameric pyridoxal 5'-phosphate dependent enzyme, catalyzes the reversible hydrolytic cleavage of L-tyrosine to phenol and ammonium pyruvate. Here we describe the crystal structure of the Citrobacter freundii holoenzyme at 1.9 Å resolution. The structure reveals a network of protein interactions with the cofactor, pyridoxal 5'-phosphate, and details of coordination of the catalytically important K+ ion. We also present the structure of the apoenzyme at 1.85 Å ; ; resolution. Both structures were determined using crystals grown at pH 8.0, which is close to the pH of the maximal enzymatic activity (8.2). Comparison of the apoenzyme structure with the one previously determined at pH 6.0 reveals significant differences. The data suggest that the decrease of the enzymatic activity at pH 6.0 may be caused by conformational changes in the active site residues Tyr71, Tyr291, and Arg381 and in the monovalent cation binding residue Glu69. Moreover, at pH 8.0 we observe two different active site conformations: open, which was characterized before, and closed, which is observed for the first time in beta-eliminating lyases. In the closed conformation a significant part of the small domain undergoes an extraordinary motion of up to 12 Å ; ; toward the large domain, closing the active site cleft and bringing the catalytically important Arg381 and Phe448 into the active site. The closed conformation allows rationalization of the results of previous mutational studies and suggests that the observed active site closure is critical for the course of the enzymatic reaction and for the enzyme’ s specificity toward its physiological substrate. Finally, the closed conformation allows us to model keto(imino)quinonoid, the key transition intermediate.

tyrosine phenol-lyase; PLP-dependent enzymes; conformational flexibility; active site closure

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Podaci o izdanju

45 (24)

2006.

7544-7552

objavljeno

0006-2960

10.1021/bi0601858

Povezanost rada

Kemija

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