Catalytically Important Amino Acid Residues in Endoglucanases from a Mutant Strain Trichoderma sp. M₇

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S. D. Petrova^*, N. G. Bakalova, and D. N. Kolev

Department of Biochemistry, Faculty of Biology, University of Sofia “St. Kliment Ohridski”, Dragan Tsankov Blvd. 8, 1164 Sofia, Bulgaria; fax: +3592-8656641; E-mail: spetrova@biofac.uni-sofia.bg

^* To whom correspondence should be addressed.

Received October 19, 2004; Revision received December 16, 2004

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Two endoglucanases, EG-III (49.7 kD) and EG-IV (47.5 kD), from a mutant strain Trichoderma sp. M₇ were modified with several specific reagents. Water-soluble carbodiimide completely inactivated only one of the purified endoglucanases and kinetic analysis indicated that at least two molecules of carbodiimide bind to EG-IV for inactivation. The reaction followed pseudo-first-order kinetics with a second-order rate constant of 3.57*10^-5 mM^-1*min^-1. Both endoglucanases were inhibited by iodoacetamide, but the absence of substrate protection excluded direct involvement of cysteine residues in the catalysis. N-Bromosuccinimide (NBS) showed a strong inhibitory effect on both endoglucanases, suggesting that tryptophan residues are essential for the activity and binding to the substrate, since the presence of substrates or analogs prior to NBS modification protected the enzymes against inactivation.

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KEY WORDS: cellulase, chemical modification, endoglucanase, Trichoderma

DOI: 10.1134/S0006297906130049

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