Reaction mechanisms of Trp120→Phe and wild-type glucoamylases from Aspergillus niger. Interactions with maltooligodextrins and acarbose

K. Olsen, U. Christensen, Michael Sierks, B. Svensson

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

Interactions of wild-type and Trp120→Phe glucoamylase with maltooligodextrin (G(x)) substrates and the tight-binding inhibitor acarbose (A) were investigated here using stopped-flow fluorescence spectroscopy and steady-state kinetic measurements. All wild-type and Trp120→Phe glucoamylase reactions followed the three-step model E + G(x)(or A) (k1) ⇆ (k-1) EG(x)(or A) (k2) ⇆ (k-2) E*G(x)(or A) (k3) → E + P or E-A, previously shown to account for the glucoamylase-maltose system [Olsen, K., Svensson, B., and Christensen, U. (1992) Eur. J. Biochem. 209, 777-784]. K1 = k- 1/k1, k2, and k-2, and the catalytic constant, k3, are determined. Binding of maltooligodextrins in the first reaction step is weak, with little difference between wild-type and Trp120→Phe glucoamylase. The second step, involving a conformational change, in contrast, is strongly influenced by the mutation and by the substrate length. Here wild-type glucoamylase reacts faster and forms more stable intermediates the longer the substrate. In contrast, Trp120→Phe reacts slower the longer the substrate. The effect of the mutation is thus smallest on maltose. The Trp120→Phe substitution reduces the fluorescence signal only by 12-20%, indicating that other tryptophanyl residues are important in reporting the conformational change. Trp120 also strongly influences the actual catalytic step, since the mutation decreases the k(c) values 30-80-fold. Acarbose behaves similar to maltotetraose in the first and the second steps with wild-type but not the Trp120→Phe glucoamylase. Also, a third step in the acarbose reaction which parallels the catalytic step is strongly affected by the mutation. The rate constant k3 increases 200-fold.

Original languageEnglish (US)
Pages (from-to)9686-9693
Number of pages8
JournalBiochemistry
Volume32
Issue number37
StatePublished - 1993
Externally publishedYes

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Acarbose
Glucan 1,4-alpha-Glucosidase
Aspergillus niger
Aspergillus
Mutation
Maltose
Substrates
Fluorescence Spectrometry
Fluorescence spectroscopy
Rate constants
Substitution reactions
Fluorescence
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reaction mechanisms of Trp120→Phe and wild-type glucoamylases from Aspergillus niger. Interactions with maltooligodextrins and acarbose. / Olsen, K.; Christensen, U.; Sierks, Michael; Svensson, B.

In: Biochemistry, Vol. 32, No. 37, 1993, p. 9686-9693.

Research output: Contribution to journalArticle

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title = "Reaction mechanisms of Trp120→Phe and wild-type glucoamylases from Aspergillus niger. Interactions with maltooligodextrins and acarbose",
abstract = "Interactions of wild-type and Trp120→Phe glucoamylase with maltooligodextrin (G(x)) substrates and the tight-binding inhibitor acarbose (A) were investigated here using stopped-flow fluorescence spectroscopy and steady-state kinetic measurements. All wild-type and Trp120→Phe glucoamylase reactions followed the three-step model E + G(x)(or A) (k1) ⇆ (k-1) EG(x)(or A) (k2) ⇆ (k-2) E*G(x)(or A) (k3) → E + P or E-A, previously shown to account for the glucoamylase-maltose system [Olsen, K., Svensson, B., and Christensen, U. (1992) Eur. J. Biochem. 209, 777-784]. K1 = k- 1/k1, k2, and k-2, and the catalytic constant, k3, are determined. Binding of maltooligodextrins in the first reaction step is weak, with little difference between wild-type and Trp120→Phe glucoamylase. The second step, involving a conformational change, in contrast, is strongly influenced by the mutation and by the substrate length. Here wild-type glucoamylase reacts faster and forms more stable intermediates the longer the substrate. In contrast, Trp120→Phe reacts slower the longer the substrate. The effect of the mutation is thus smallest on maltose. The Trp120→Phe substitution reduces the fluorescence signal only by 12-20{\%}, indicating that other tryptophanyl residues are important in reporting the conformational change. Trp120 also strongly influences the actual catalytic step, since the mutation decreases the k(c) values 30-80-fold. Acarbose behaves similar to maltotetraose in the first and the second steps with wild-type but not the Trp120→Phe glucoamylase. Also, a third step in the acarbose reaction which parallels the catalytic step is strongly affected by the mutation. The rate constant k3 increases 200-fold.",
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