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Role of Lysine-256 in Citrobacter freundii tyrosine phenol-lyase in monovalent cation activation
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2004 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 45, 14412-14419 p.Article in journal (Refereed) Published
Abstract [en]

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K+ or NH4+, for high activity. We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation [Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R. S. (2000) Biochemistry 39, 8546-8555]. Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation. This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase. We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation. K256A and K256H TPLs have low activity (k(cat)/K-m values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor. In contrast, K256R TPL has higher activity (k(cat)/K-m about 10% of wild-type TPL), is activated by K+, and exhibits fluorescence emission from the PLP cofactor. K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL. E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation. This mutant TPL has wild-type activity with S-Et-L-CYS or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr. E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence. In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain I mol of PLP per subunit. Thus, E69D/K256R TPL appears to have altered dynamics. All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates. Thus, Lys-256 is not the base which removes the alpha-proton during catalysis. The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.

Place, publisher, year, edition, pages
2004. Vol. 43, no 45, 14412-14419 p.
Keyword [en]
Binding energy, Catalysis, Chemical activation, Enzyme inhibition, Fluorescence, Hydrogen bonds, Phenols, Positive ions
National Category
Biochemistry and Molecular Biology
URN: urn:nbn:se:kth:diva-40295DOI: 10.1021/bi0484062ISI: 000225095000012ScopusID: 2-s2.0-8544266044OAI: diva2:441437
QC 20110916Available from: 2011-09-16 Created: 2011-09-14 Last updated: 2011-09-16Bibliographically approved

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Tavakoli, Khadijeh
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