Role of Lysine-256 in Citrobacter freundii Tyrosine Phenol-lyase in Monovalent Cation Activation

Abstract

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.

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

“…This water molecule lies in the active site, and it is hydrogen-bonded by the PLP phosphate group and hydroxyl groups from side chains of the active site residues Ser51, Tyr71, and Tyr291 (Figure 3). These concatenated interactions of the cation-binding site with the active site residues demonstrate the critical role of Lys256 in the formation of the monovalent cation binding site in agreement with previous biochemical observations (47) and provide an insight into the influence of monovalent cations on the activity of TPL (48,49). At cation concentrations lower than 100 mM, K + , NH 4 + , and Rb + were shown to be the most effective activators, Cs + and Li + were significantly less effective, while the effect of Na + was negligible.…”

The role of substrate strain in the mechanism of the carbon–carbon lyases

“…A unique feature of TIL and TPL is another strictly conserved lysine (Lys-256 in C. freundii TPL) immediately preceding the PLP-binding lysine, and hydrogen bonded to a water molecule bound to the MVC. This lysine appears to play a role in the activation of these enzymes by MVCs [24]. The residues that contact the substrate directly include a conserved tyrosine (Tyr- (Fig.…”

Inactivation of tyrosine phenol‐lyase by Pictet–Spengler reaction and alleviation by T15A mutation on intertwined N‐terminal arm

“…The network of hydrogen bonds and salt bridges formed upon the binding of PLP is known to influence the quaternary structure of tryptophanases [17]. Therefore, when considering the common structural features of α‐family PLP enzymes [26,27], the T15A mutation on the N‐terminal arm may have increased the rigidity of the cofactor binding architecture of C. freundii TPL through adjusting the quaternary interfaces. One possible communication between the N‐terminal arm and the active site is through Tyr71, which belongs to the adjacent subunit of the catalytic dimer.…”