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Structural Studies of the Enzymatic Mechanism of Citrobacter freundii Tyrosine Phenol-lyase

Tyrosine phanol-lyase (TPL) catalyzes the reversible hydrolytic beta-elimination reaction of L-tyrosine to produce phenol and ammonium pyruvate. It is a homotetrameric protein which belongs to the Fold Type I (aspartate aminotransferase) family of the PLP-dependent enzymes (1). The active sites (one per subunit) are situated at the interface of two subunits forming the so-called catalytic dimer. Each protein subunit and the corresponding active site of Citrobacter freundii TPL can possess two different conformations: open and closed (2). The closed conformation is formed from the open one by the rotation of the small rigid region by 16° about a hinge connecting it with the large rigid region. Our crystallographic studies (3) have showed that the active-site closure protects the key quinonoid intermediate from the solvent and brings the catalytically important residues Arg381, Thr124 and Phe448 into positions suitable for the interaction with the substrate thus facilitating the abstraction of phenol during the beta-elimination of L-tyrosine. References 1. Antson, A. A., Demidkina, T. V., Gollnick, P., Dauter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H., and Wilson, K. S., Biochemistry, 32, 4195– 4206 (1993). 2. Milić, D., Matković-Čalogović, D., Demidkina, T. V., Kulikova, V. V., Sinitzina, N. I., and Antson, A. A., Biochemistry 45, 7544– 7552 (2006). 3. Milić, D., Demidkina, T. V., Faleev, N. G., Matković-Čalogović, D., and Antson, A. A., J. Biol. Chem., In press, (2008), doi:10.1074/jbc.M802061200.

tyrosine phenol-lyase; beta-elimination; quinonoid intermediate; closed conformation; pyridoxal 5'-phosphate; structural enzymology; X-ray structural analysis

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