Role of Arginine 226 in the Mechanism of Tryptophan Indole-Lyase from Proteus vulgaris

V. V. Kulikova¹, L. N. Zakomirdina¹, N. P. Bazhulina¹, I. S. Dementieva¹, N. G. Faleev², P. D. Gollnick³, and T. V. Demidkina^1*

¹Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow 119991, Russia; fax: (7-095) 135-1405; E-mail: tvd@eimb.ru

²Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, ul. Vavilova 28, Moscow 117813, Russia; fax: (7-095) 135-5085; E-mail: ngfal@ineos.ac.ru

³Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260, USA; E-mail: gollnick@acsu.buffalo.edu

^* To whom correspondence should be addressed.

Received May 27, 2003

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In the spatial structure of tryptophanase from Proteus vulgaris the guanidinium group of arginine 226 forms a salt bridge with the 3´-oxygen atom of the coenzyme. The replacement of arginine 226 with alanine using site-directed mutagenesis reduced the affinity of the coenzyme for the protein by one order of magnitude compared to the wild-type enzyme. The catalytic activity of the mutant enzyme in the reaction with L-tryptophan was reduced 10⁵-fold compared to the wild-type enzyme. The rates of the reactions with some other substrates decreased 10³-10⁴-fold. The mutant enzyme catalyzed exchange of the C-alpha-proton in complexes with some inhibitors with rates reduced 10²-fold compared to the wild-type enzyme. Absorption and circular dichroism spectra of the mutant enzyme and the enzyme-inhibitor complexes demonstrate that the replacement of arginine 226 with alanine does not significantly affect the tautomeric equilibrium of the internal aldimine, but it leads to an alteration of the optimal conformation of the coenzyme-substrate intermediates.

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KEY WORDS: tryptophan indole-lyase from Proteus vulgaris, pyridoxal 5´-phosphate, arginine 226, site-directed mutagenesis

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