Site‐Directed Mutagenesis of His343→Ala in Citrobacter freundii Tyrosine Phenol‐Lyase

His343 in Citrobacter freundii tyrosine phenol-lyase is conserved in all known sequences of both tyrosine phenol-lyase and tryptophan indole-lyase; it is located near the active-site Lys257 in C. freundii tyrosine phenol-lyase [Antson, A. A., Demidkina, T. V., Gollnick, P., Danter, Z., Von Tersch, R. L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H. & Wilson, K. S. (1993) Biochemistry 32, 4195–4206]. In order to evaluate the role of His343 in the reaction mechanism of tyrosine phenol-lyase and tryptophan indolelyase, we have mutated it to Ala; the former mutant is referred to as [H343A]tyrosine phenol lyase. All substrates for α,/β-elimination (except S -ethyl-l-cysteine) exhibited lower kcat (10–30%) and kcat/Km (1–10%) values with [H343A]tyrosine phenol-lyase than with the wild-type enzyme. The mutant also shows slower rates of deuterium isotope exchange for l-phenylalanine and l-methionine than does the wild type. The pH-dependent behavior in the reaction of 3-fluoro-l-tyrosine with wild-type tyrosine phenol-lyase is identical to that of l-tyrosine described previously [Kiick, D. M. & Phillips, R. S. (1988) Biochemistry 27, 7333–7338]. The pH profile of kcatKm for this reaction exhibits two pKa values with an average of 7.7 ± 0.2, indicating that the catalytic mechanism requires two essential basic groups. The pH profile of kcat/Km for 3-fluoro-L-tyrosine with [H343A]tyrosine phenol-lyase also exhibits two pKa values with an average of 7.8 ± 0.3. However, kcat for 3-fluoro-l-tyrosine is pH-dependent for the mutant, exhibiting two pKa values with an average of about 7.8, whereas it is pH-independent for the wild type. Steady-state kinetic isotope effects on the reactions with wild-type and [H343A]tyrosine phenol-lyase were examined at various pH values. For the wild type, the values of the isotope effects on fccat and kcat/Km for 3-fluoro-l-[α-2H]-tyrosine are independent of pH and equal to 3.9 ± 0.2 and 2.2±0.3, respectively, while the corresponding values for [H343A]tyrosine phenol-lyase are 5.4 ± 0.2 and 3.8 ± 0.3, respectively. Based on these observations and the pH data, we conclude that α-proton abstraction is apparently the rate-determining step in the reaction of 3-fluoro-l-tyrosine with [H343A]tyrosine phenol-lyase; however, quinonoid intermediate formation (140 s −1) is much faster than kcat of 3-fluoro-L-tyrosine (1.4 s −1) for wild-type tyrosine phenol-lyase; thus, the mutation of His343Ala has apparently changed the rate-determining step of the reaction with 3-fluoro-l-tyrosine. l-Methionine, l-homoserine, l-phenylalanine and l-alanine form quinonoid intermediates with wild-type tyrosine phenol-lyase which exhibit absorbance peaks at about 500 nm but the intensity of these absorbance peaks is dramatically reduced for the mutant. Single-wavelength stopped-flow kinetic measurements demonstrate that [H343A]tyrosine phenol-lyase exhibits slower rate constants for α-deprotonation and faster rate constants for reprotonation than does the wild-type. Taken together, these results demonstrate that, while His343 is not an essential basic group in tyrosine phenol-lyase, it does play an important function in catalysis, possibly by facilitating the conformational change from an ‘open’ to ‘closed’ form when substrates bind.