Site-directed mutagenesis of the catalytic base glutamic acid 400 in glucoamylase from Aspergillus niger and of tyrosine 48 and glutamine 401, both hydrogen-bonded to the γ-carboxylate group of glutamic acid 400

T. P. Frandsen, C. Dupont, J. Lehmbeck, B. Stoffer, Michael Sierks, R. B. Honzatko, B. Svensson

Research output: Contribution to journalArticle

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Abstract

Replacement of the catalytic base Glu400 by glutamine in glucoamylase from Aspergillus niger affects both substrate ground-state binding and transition- state stabilization. Compared to those of the wild-type enzyme, K(m) values for maltose and maltoheptaose are 12- and 3-fold higher for the Glu400→Gln mutant, with k(cat) values 35- and 60-fold lower, respectively, for the same substrates. This unusually high residual activity for a glycosylase mutant at a putative catalytic group is tentatively explained by a reorganization of the hydrogen bond network, using the crystal structure of the related Aspergillus awamori var. X100 glucoamylase in complex with 1-deoxynojirimycin [Harris, E. M. S., Aleshin, A. E., Firsov, L. M., and Honzatko, R. B. (1993) Biochemistry 32, 1618-1626]. Supposedly Gln400 in the mutant hydrogen bonds to the invariant Tyr48, as does Glu400 in the wild-type enzyme. For Tyr48→Trp A. niger glucoamylase k(cat) is reduced 80-100-fold, while K(m) is increased only 2-3-fold. Gln401 also hydrogen bonds to Glu400, but its mutation to glutamic acid has only a minor effect on activity. The Tyr48→Trp and Glu400→Gln glucoamylases share particular features in displaying unusually high activity below pH 4.0-which reflects lack of the wild-type catalytic base function-and unusually low binding affinity at subsite 2. Both mutants have lost 13-16 kJ mol-1 in transition-state stabilization energy. The Glu400→Gln mutant confirms the role of Glu400 in catalysis, and mutation of Tyr48 suggests that this side chain is functionally linked to Glu400 and is important for maintaining the active site geometry and for stabilization of an oxocarbonium ion substrate intermediate. The properties of the glucoamylase mutants are compared with results of mutational analysis in other carbohydrases.

Original languageEnglish (US)
Pages (from-to)13808-13816
Number of pages9
JournalBiochemistry
Volume33
Issue number46
DOIs
StatePublished - 1994
Externally publishedYes

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Glucan 1,4-alpha-Glucosidase
Mutagenesis
Aspergillus niger
Aspergillus
Site-Directed Mutagenesis
Glutamine
Tyrosine
Glutamic Acid
Hydrogen
Hydrogen bonds
Stabilization
Substrates
1-Deoxynojirimycin
Mutation
Biochemistry
Maltose
Enzymes
Electron transitions
Catalysis
Ground state

ASJC Scopus subject areas

  • Biochemistry

Cite this

Site-directed mutagenesis of the catalytic base glutamic acid 400 in glucoamylase from Aspergillus niger and of tyrosine 48 and glutamine 401, both hydrogen-bonded to the γ-carboxylate group of glutamic acid 400. / Frandsen, T. P.; Dupont, C.; Lehmbeck, J.; Stoffer, B.; Sierks, Michael; Honzatko, R. B.; Svensson, B.

In: Biochemistry, Vol. 33, No. 46, 1994, p. 13808-13816.

Research output: Contribution to journalArticle

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title = "Site-directed mutagenesis of the catalytic base glutamic acid 400 in glucoamylase from Aspergillus niger and of tyrosine 48 and glutamine 401, both hydrogen-bonded to the γ-carboxylate group of glutamic acid 400",
abstract = "Replacement of the catalytic base Glu400 by glutamine in glucoamylase from Aspergillus niger affects both substrate ground-state binding and transition- state stabilization. Compared to those of the wild-type enzyme, K(m) values for maltose and maltoheptaose are 12- and 3-fold higher for the Glu400→Gln mutant, with k(cat) values 35- and 60-fold lower, respectively, for the same substrates. This unusually high residual activity for a glycosylase mutant at a putative catalytic group is tentatively explained by a reorganization of the hydrogen bond network, using the crystal structure of the related Aspergillus awamori var. X100 glucoamylase in complex with 1-deoxynojirimycin [Harris, E. M. S., Aleshin, A. E., Firsov, L. M., and Honzatko, R. B. (1993) Biochemistry 32, 1618-1626]. Supposedly Gln400 in the mutant hydrogen bonds to the invariant Tyr48, as does Glu400 in the wild-type enzyme. For Tyr48→Trp A. niger glucoamylase k(cat) is reduced 80-100-fold, while K(m) is increased only 2-3-fold. Gln401 also hydrogen bonds to Glu400, but its mutation to glutamic acid has only a minor effect on activity. The Tyr48→Trp and Glu400→Gln glucoamylases share particular features in displaying unusually high activity below pH 4.0-which reflects lack of the wild-type catalytic base function-and unusually low binding affinity at subsite 2. Both mutants have lost 13-16 kJ mol-1 in transition-state stabilization energy. The Glu400→Gln mutant confirms the role of Glu400 in catalysis, and mutation of Tyr48 suggests that this side chain is functionally linked to Glu400 and is important for maintaining the active site geometry and for stabilization of an oxocarbonium ion substrate intermediate. The properties of the glucoamylase mutants are compared with results of mutational analysis in other carbohydrases.",
author = "Frandsen, {T. P.} and C. Dupont and J. Lehmbeck and B. Stoffer and Michael Sierks and Honzatko, {R. B.} and B. Svensson",
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TY - JOUR

T1 - Site-directed mutagenesis of the catalytic base glutamic acid 400 in glucoamylase from Aspergillus niger and of tyrosine 48 and glutamine 401, both hydrogen-bonded to the γ-carboxylate group of glutamic acid 400

AU - Frandsen, T. P.

AU - Dupont, C.

AU - Lehmbeck, J.

AU - Stoffer, B.

AU - Sierks, Michael

AU - Honzatko, R. B.

AU - Svensson, B.

PY - 1994

Y1 - 1994

N2 - Replacement of the catalytic base Glu400 by glutamine in glucoamylase from Aspergillus niger affects both substrate ground-state binding and transition- state stabilization. Compared to those of the wild-type enzyme, K(m) values for maltose and maltoheptaose are 12- and 3-fold higher for the Glu400→Gln mutant, with k(cat) values 35- and 60-fold lower, respectively, for the same substrates. This unusually high residual activity for a glycosylase mutant at a putative catalytic group is tentatively explained by a reorganization of the hydrogen bond network, using the crystal structure of the related Aspergillus awamori var. X100 glucoamylase in complex with 1-deoxynojirimycin [Harris, E. M. S., Aleshin, A. E., Firsov, L. M., and Honzatko, R. B. (1993) Biochemistry 32, 1618-1626]. Supposedly Gln400 in the mutant hydrogen bonds to the invariant Tyr48, as does Glu400 in the wild-type enzyme. For Tyr48→Trp A. niger glucoamylase k(cat) is reduced 80-100-fold, while K(m) is increased only 2-3-fold. Gln401 also hydrogen bonds to Glu400, but its mutation to glutamic acid has only a minor effect on activity. The Tyr48→Trp and Glu400→Gln glucoamylases share particular features in displaying unusually high activity below pH 4.0-which reflects lack of the wild-type catalytic base function-and unusually low binding affinity at subsite 2. Both mutants have lost 13-16 kJ mol-1 in transition-state stabilization energy. The Glu400→Gln mutant confirms the role of Glu400 in catalysis, and mutation of Tyr48 suggests that this side chain is functionally linked to Glu400 and is important for maintaining the active site geometry and for stabilization of an oxocarbonium ion substrate intermediate. The properties of the glucoamylase mutants are compared with results of mutational analysis in other carbohydrases.

AB - Replacement of the catalytic base Glu400 by glutamine in glucoamylase from Aspergillus niger affects both substrate ground-state binding and transition- state stabilization. Compared to those of the wild-type enzyme, K(m) values for maltose and maltoheptaose are 12- and 3-fold higher for the Glu400→Gln mutant, with k(cat) values 35- and 60-fold lower, respectively, for the same substrates. This unusually high residual activity for a glycosylase mutant at a putative catalytic group is tentatively explained by a reorganization of the hydrogen bond network, using the crystal structure of the related Aspergillus awamori var. X100 glucoamylase in complex with 1-deoxynojirimycin [Harris, E. M. S., Aleshin, A. E., Firsov, L. M., and Honzatko, R. B. (1993) Biochemistry 32, 1618-1626]. Supposedly Gln400 in the mutant hydrogen bonds to the invariant Tyr48, as does Glu400 in the wild-type enzyme. For Tyr48→Trp A. niger glucoamylase k(cat) is reduced 80-100-fold, while K(m) is increased only 2-3-fold. Gln401 also hydrogen bonds to Glu400, but its mutation to glutamic acid has only a minor effect on activity. The Tyr48→Trp and Glu400→Gln glucoamylases share particular features in displaying unusually high activity below pH 4.0-which reflects lack of the wild-type catalytic base function-and unusually low binding affinity at subsite 2. Both mutants have lost 13-16 kJ mol-1 in transition-state stabilization energy. The Glu400→Gln mutant confirms the role of Glu400 in catalysis, and mutation of Tyr48 suggests that this side chain is functionally linked to Glu400 and is important for maintaining the active site geometry and for stabilization of an oxocarbonium ion substrate intermediate. The properties of the glucoamylase mutants are compared with results of mutational analysis in other carbohydrases.

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