Catalytic mechanism of glucoamylase probed by mutagenesis in conjunction with hydrolysis of α-D-glucopyranosyl fluoride and maltooligosaccharides

Michael R. Sierks, Birte Svensson

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20 Scopus citations

Abstract

The catalytic mechanism of glucoamylase (GA) is investigated by comparing kinetic results obtained using α-D-glucosyl fluoride (GF) and maltooligosaccharides as substrates for wild-type and four active site mutant GAs, Tyr116→Ala, Trp120→Phe, Asp176→Asn, and Glu400→Gln. These replacements decreased the activity (k(cat)/K(M)) toward maltose by 6-320- fold. Toward GF, however, Tyr116→Ala and Trp120→Phe GAs, showed wild-type and twice wild-type level activity, while Asp176→Asn and Glu400→Gln GAs had 22- and 665-fold lower activity, respectively. Glu400, the catalytic base, is suggested to strengthen ground-state binding in subsite 1, and Asp176 does so at subsites 1 and 2. Tyr116 and Trp120 belong to an aromatic cluster that is slightly removed from the catalytic site and not critical for GF hydrolysis, but which is probably involved in maltooligosaccharide transition-state stabilization. Since the mutation of groups near the catalytic site decreased activity for both GF and maltose, but substitution of Tyr116 and Trp120 decreased activity only for maltose, interaction with the substrate aglycon part may be implicated in the rate-limiting step. Rate-limiting aglycon product release was suggested previously for GA-catalyzed hydrolysis [Kitahata, S., Brewer, C. F., Genghof, D. S., Sawai, T., and Hehre, E. H. (1981) J. Biol. Chem. 256, 6017-6026]. For Glu400→Gln and wild-type GA complexed with GF, the pH-activity (k(cat)) profile shows a pK(a) of 2.8. When these two enzymes were complexed with maltose, however, only wild-type GA had a titrating base group, assigned to Glu400 [Frandsen, T. P., Dupont, C., Lehmbeck, J., Stoffer, B., Sierks, M. R., Honzatko, R. B., and Svensson, B. (1994) Biochemistry 33, 13808-13816]. Thus, GF binding to Glu40→Gln GA presumably elicits the deprotonation of a carboxyl group that facilitates catalysis.

Original languageEnglish (US)
Pages (from-to)1865-1871
Number of pages7
JournalBiochemistry
Volume35
Issue number6
DOIs
StatePublished - Feb 13 1996
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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