Formation in vitro of hybrid dimers of H463F and Y74F mutant Escherichia coli tryptophan indole-lyase rescues activity with L-tryptophan

Biochemistry
R S PhillipsAjith V Kamath

Abstract

Y74F and H463F mutant forms of Escherichia coli tryptophan indole-lyase (Trpase) have been prepared. These mutant proteins have very low activity with L-Trp as substrate (kcat and kcat/Km values less than 0.1% of wild-type Trpase). In contrast, these mutant enzymes exhibit much higher activity with S-(o-nitrophenyl)-L-cysteine and S-ethyl-L-cysteine (kcat/Km values about 1-50% of wild-type Trpase). Thus, Tyr-74 and His-463 are important for the substrate specificity of Trpase for L-Trp. H463F Trpase is not inhibited by a potent inhibitor of wild-type Trpase, oxindolyl-L-alanine, and does not exhibit the pK(a) of 6.0 seen in previous pH dependence studies [Kiick, D. M., and Phillips, R. S. (1988) Biochemistry 27, 7333]. These results suggest that His-463 may be the catalytic base with a pK(a) of 6.0 and Tyr-74 may be a general acid catalyst for the elimination step, as we found previously with tyrosine phenol-lyase [Chen, H., Demidkina, T. V., and Phillips, R. S. (1995) Biochemistry 34, 12776]. H463F Trpase reacts with L-Trp and S-ethyl-L-cysteine in rapid-scanning stopped-flow experiments to form equilibrating mixtures of external aldimine and quinonoid intermediates, similar to those observed with wild-type Trpase. In contrast...Continue Reading

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Citations

Apr 11, 2003·Biochimica Et Biophysica Acta·Robert S PhillipsNicolai G Faleev
Jul 19, 2014·Bioorganic Chemistry·Robert S PhillipsNicolai G Faleev
Nov 18, 2010·Archives of Biochemistry and Biophysics·Robert S PhillipsDouglas Bartlett
Sep 26, 2014·Applied Biochemistry and Biotechnology·Taiyeebah NuidateVaraporn Vuddhakul
Aug 8, 2018·Acta Crystallographica. Section D, Structural Biology·Robert S PhillipsZachary A Wood
Apr 6, 2002·The Journal of Biological Chemistry·Robert S PhillipsAndrea Mozzarelli

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