PMID:11900544

Phillips, RS, Johnson, N and Kamath, AV (2002) Formation in vitro of hybrid dimers of H463F and Y74F mutant Escherichia coli tryptophan indole-lyase rescues activity with L-tryptophan.Biochemistry 41:4012-9

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 to the results with wild-type Trpase, the addition of benzimidazole to reactions of H463F Trpase with L-Trp does not result in the formation of an aminoacrylate intermediate. However, addition of benzimidazole with S-ethyl-L-cysteine results in the formation of an aminoacrylate intermediate, with lambda(max) at 345 nm, as was seen previously with wild-type Trpase [Phillips, R. S. (1991) Biochemistry 30, 5927]. This suggests that His-463 plays a specific role in the elimination step of the reaction of L-Trp. Refolding of equimolar mixtures of H463F and Y74F Trpase after unfolding in 4 M guanidine hydrochloride results in a dramatic increase in activity with L-Trp, indicating the formation of a hybrid H463F/Y74F dimer with one normal active site.

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