The role of glutamic acid-69 in the activation of Citrobacter freundii tyrosine phenol-lyase by monovalent cations

Tyrosine phenol-lyase (TPL) from Citrobacter freundii is activated about 30 -fold by monovalent cations, the most effective being K+, NH4+, and Rb+. Pr evious X-ray crystal structure analysis has demonstrated that the monovalen t cation binding site is located at the interface between subunits, with Li gands contributed by the carbonyl oxygens of Gly52 and Asn262 from one chai n and monodentate Ligation by one of the epsilon-oxygens of Glu69 from anot her chain [Antson, A. A., Demidkina, T. V., Gollnick, P., Dauter, Z., Von T ersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H., and Wilson, K. S. (1993) Biochemistry 32, 4195]. We have studied the ef fect of mutation of Glu69 to glutamine (E69Q) and aspartate (E69D) to deter mine the role of Glu69 in the activation of TPL. E69Q TPL is activated by K +, NH4+, and Rb+, with K-D values similar to wild-type TPL, indicating that the negative charge on Glu69 is not necessary for cation binding and activ ation. In contrast, E69D TPL exhibits very low basal activity and only weak activation by monovalent cations, even though monovalent cations are capab le of binding, indicating that the geometry of the monovalent cation bindin g site is critical for activation. Rapid-scanning stopped-flow kinetic stud ies of wild-type TPL show that the activating effect of the cation is seen in an acceleration of rates of quinonoid intermediate formation (30-50-fold ) and of phenol elimination. Similar rapid-scanning stopped-flow results we re obtained with E69Q TPL; however, E69D TPL shows only a 4-fold increase i n the rate of quinonoid intermediate formation with K+ Preincubation of TPL with monovalent cations is necessary to observe the rate acceleration in s topped flow kinetic experiments, suggesting that the activation of TPL by m onovalent cations is a slow process. In agreement with this conclusion, a s low increase (k < 0.5 s(-1)) in fluorescence intensity (lambda(ex) = 420 nm , lambda(em) = 505 nm) is observed when wild-type and E69Q TPL are mixed wi th K+, Rb+, and NH4+ but not Li+ or Na+. E69D TPL shows no change in fluore scence under these conditions. High concentrations (>100 mM) of all monoval ent cations result in inhibition of wild-type TPL. This inhibition is proba bly due to cation binding to the ES complex to form a complex that releases pyruvate slowly.