Replacement of lysine 269 by arginine in Escherichia coli tryptophan indole-lyase affects the formation and breakdown of quinonoid complexes

Lysine 269 in Escherichia coli tryptophan indolelyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits k_cat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while k_cat/K_m values are reduced to 2% or less. The pH profile of k_cat/ K_m for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK_a values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK_a values of 6.0 and 7.8. The pK_a for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK_a of 7.4. Steady-state kinetic isotope effects on the reaction of [α-²H]S-benzyl-L-cysteine with the K269R mutant enzyme (^Dk_cat = 2.0; ^D(k_cat/K_m) = 3.9) are larger than those of the wild-type enzyme (^Dk_cat = 1.4; ^D(k_cat/K_m) = 2.9). Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxindolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the α-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (^Dk = 4.2) than for the wild-type (^Dk = 7.9). The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme. These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.