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
References
Sep 1, 1977·Journal of Biochemistry·T Watanabe, E E Snell
Jan 1, 1979·Methods in Enzymology·W W Cleland
Jul 15, 1976·FEBS Letters·C H SuelterE E Snell
Oct 27, 1992·Biochemistry·M PookanjanatavipD V Santi
Feb 19, 1991·Biochemistry·E M Woolridge, S E Rokita
Jun 18, 1991·Biochemistry·R S Phillips
Sep 18, 1990·Biochemistry·R S PhillipsM F Dunn
Jul 1, 1987·Archives of Biochemistry and Biophysics·R S Phillips
Sep 20, 1988·Biochemistry·D M Kiick, R S Phillips
Jan 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·S R Wente, H K Schachman
Sep 1, 1981·Journal of Bacteriology·M C Deeley, C Yanofsky
Dec 4, 1984·Biochemistry·R S PhillipsL A Cohen
Oct 1, 1981·European Journal of Biochemistry·T NihiraS Fukui
Feb 1, 1995·Journal of Biochemistry·T Mizobata, Y Kawata
Feb 1, 1994·European Journal of Biochemistry·F W AlexanderP Christen
Jun 8, 1993·Biochemistry·K E Tobias, C Kahana
Jul 9, 1996·Biochemistry·D Tan, G C Ferreira
Nov 22, 1996·The Journal of Biological Chemistry·C KangH J Fromm
Dec 17, 1996·Biochemistry·M J Sloan, R S Phillips
May 27, 1997·Biochemistry·B SundararajuK S Wilson
Jan 9, 1998·Journal of Bacteriology·K MartinM Golomb
Apr 16, 1998·Biochemistry·H IkushiroH Kagamiyama
Apr 29, 1998·Journal of Molecular Biology·M N IsupovE H Harutyunyan
Jun 20, 1998·The Journal of Biological Chemistry·A S Tarun, A Theologis
Aug 22, 2000·Nature·J F HeidelbergC M Fraser
Aug 1, 1962·Proceedings of the National Academy of Sciences of the United States of America·W A NEWTON, E E SNELL
Citations
Apr 11, 2003·Biochimica Et Biophysica Acta·Robert S PhillipsNicolai G Faleev
Jun 14, 2006·Biochemistry·Dalibor MilićAlfred A Antson
Apr 20, 2002·The Biochemical Journal·Tatyana V DemidkinaRobert S Phillips
Jun 29, 2010·Isotopes in Environmental and Health Studies·Elzbieta WinnickaMarianna Kanska
Jul 19, 2014·Bioorganic Chemistry·Robert S PhillipsNicolai G Faleev
Nov 18, 2010·Archives of Biochemistry and Biophysics·Robert S PhillipsDouglas Bartlett
Feb 27, 2013·The FEBS Journal·Austin P Harris, Robert S Phillips
Feb 4, 2006·Biochimica Et Biophysica Acta·Vitalia V KulikovaNicolai G Faleev
Sep 26, 2014·Applied Biochemistry and Biotechnology·Taiyeebah NuidateVaraporn Vuddhakul
Aug 3, 2012·Biochemistry·Robert S PhillipsSam Hay
Aug 8, 2018·Acta Crystallographica. Section D, Structural Biology·Robert S PhillipsZachary A Wood
Dec 4, 2003·Oncogene·Irina V LebedevaPaul B Fisher
May 5, 2005·Oncogene·Yu YangXiao-Feng Yang
Apr 6, 2002·The Journal of Biological Chemistry·Robert S PhillipsAndrea Mozzarelli