Functional and structural roles of the highly conserved Trp120 loop region of glucoamylase from Aspergillus awamori

Sateesh Natarajan, Michael Sierks

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Abstract

The functional role of a loop region, highly conserved among glucoamylase and other starch hydrolases which also includes the essential Trp120 of Aspergillus awamori, is investigated. Residues 121-125 of A. awamori glucoamylase were singly substituted, and their individual effects on catalytic activity and thermal stability were determined. The Arg122→Tyr mutation displayed opposing effects for shorter and longer maltooligosaccharide substrates, K(m) decreasing for shorter substrates but increasing for longer substrates. The Pro123→Gly mutation decreases the thermal stability of glucoamylase by 19 °C with little effect on activity. The Gln124→His substitution decreases k(cat) for all substrates 10-15-fold. Gly121→Thr and Arg125→Lys had only minor effects on glucoamylase activity. While Arg122→Tyr, Gln124→His, and the previously constructed Trp120→Phe [Sierks, M. R., Svensson, B., Ford, C., and Reilly, P. J. (1989) Protein Eng. 2, 621-625] glucoamylases have significantly reduced activity toward maltose hydrolysis, all mutations in the Trp120 loop region retain wild-type level activity toward α-D-glucosyl fluoride hydrolysis. The Trp120 loop region therefore plays a major role in directing conformational changes controlling the postulated rate-limiting product release step, even though only Trp120 is indicated to interact with acarbose in the crystal structure [Aleshin, A. E., Firsoy, L. M., and Honzatko, R. B. (1994) J. Biol. Chem. 269, 15631-15639]. Side chains of residues 116, 120, 122, and 124 oriented in one direction play crucial roles in the enzyme mechanism, while side chains of residues 119, 121, 123, and 125, oriented in the opposite direction, play only minor roles.

Original languageEnglish (US)
Pages (from-to)3050-3058
Number of pages9
JournalBiochemistry
Volume35
Issue number9
DOIs
Publication statusPublished - Mar 5 1996
Externally publishedYes

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ASJC Scopus subject areas

  • Biochemistry

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