Tryptophan Residues in alpha-Galactosidase from Trichoderma reesei

A. M. Kachurin, S. V. Protasenya, K. A. Shabalin, V. V. Isaev-Ivanov, A. M. Golubev, and K. N. Neustroev^*

St. Petersburg Konstantinov Nuclear Physics Institute, Orlova roshcha, Gatchina, 188351 Russia; fax: (81271) 32-303; E-mail: neustk@omrb.pnpi.spb.ru

^* To whom correspondence should be addressed.

Received November 11, 1997; Revision received February 2, 1998

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Tryptophan residues in alpha-galactosidase were modified with bromosuccinimide. The fact that galactose, a specific inhibitor of alpha-galactosidase, does not prevent this modification demonstrates that tryptophan residues are not located in galactose binding sites. Analysis of the inactivation kinetics revealed two groups of Trp residues (8.5 and 7.5 residues) with different accessibility for N-bromosuccinimide. We studied specific quenching of alpha-galactosidase fluorescence resulting from modification of an sulfhydryl group in the active site of the enzyme with Hg²⁺ and Ag⁺ ions. The specific quenching is due to conformational changes of the enzyme. Forster’s radii were determined for various protein--chromophore complexes. Dynamic quenching of alpha-galactosidase fluorescence was investigated. To describe abnormal dynamic quenching in alpha-galactosidase, a modification of the Stern--Volmer equation is suggested.

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KEY WORDS: alpha-galactosidase, Trichoderma reesei, enzyme active site, sulfhydryl group, fluorescence spectroscopy

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