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 indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less. The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8. The pK alpha 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 alpha of 7.4. Steady-state kinetic isotope effects on the reaction of [alpha-2H]S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 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 oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the alpha-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.
Department of Chemistry, School of Chemical Sciences, University of Georgia, Athens 30602.
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RS
Phillips
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Richter
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Gollnick
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P
Brzovic
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Dunn
D001120:Arginine / Q000737:chemistry; D003545:Cysteine / Q000031:analogs & derivatives / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D011809:Quinones / Q000378:metabolism*; D014368:Tryptophanase / Q000737:chemistry / Q000378:metabolism*
D011809:Quinones; C019501:S-o-nitrophenyl-L-cysteine; D001120:Arginine; C002206:S-benzylcysteine; D014368:Tryptophanase; D008239:Lysine; D003545:Cysteine