Role of lysine-256 in Citrobacter freundii tyrosine phenol-lyase in monovalent cation activation.
Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K(+) or NH(4)(+), 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 1 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.
Phillips
Robert S
RS
Department of Chemistry, University of Georgia, Athens, Georgia 30602-2556, USA. rsphillips@chem.uga.edu
Chen
Hao Yuan
HY
Shim
David
D
Lima
Santiago
S
Tavakoli
Khadijeh
K
Sundararaju
Bakthavatsalam
B
D000409:Alanine / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002414:Cations, Monovalent / Q000378:metabolism; D016971:Citrobacter freundii / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006639:Histidine / Q000235:genetics; D008239:Lysine / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011817:Rabbits; D013053:Spectrophotometry; D014447:Tyrosine Phenol-Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D002414:Cations, Monovalent; D001224:Aspartic Acid; D018698:Glutamic Acid; D006639:Histidine; D011732:Pyridoxal Phosphate; D001120:Arginine; D014447:Tyrosine Phenol-Lyase; D008239:Lysine; D000409:Alanine; D011188:Potassium