Site-Directed Mutagenesis of His343Ala in Citrobacter freundii Tyrosine Phenol-Lyase. Effects on the Kinetic Mechanism and Rate-Determining Step

Abstract

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 indole-lyase, we have mutated it to Ala; the former mutant is referred to as [H343A]tyrosine phenol lyase. All substrates for alpha, beta-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 kcat/Km 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 kcat and kcat/Km for 3-fluoro-L-[alpha-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.(ABSTRACT TRUNCATED AT 400 WORDS)