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 language | English (US) |
|---|---|
| Pages (from-to) | 9686-9693 |
| Number of pages | 8 |
| Journal | Biochemistry |
| Volume | 32 |
| Issue number | 37 |
| State | Published - 1993 |
| Externally published | Yes |
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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 journal › Article
}
TY - JOUR
T1 - Reaction mechanisms of Trp120→Phe and wild-type glucoamylases from Aspergillus niger. Interactions with maltooligodextrins and acarbose
AU - Olsen, K.
AU - Christensen, U.
AU - Sierks, Michael
AU - Svensson, B.
PY - 1993
Y1 - 1993
N2 - 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.
AB - 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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M3 - Article
C2 - 8373772
AN - SCOPUS:0027429148
VL - 32
SP - 9686
EP - 9693
JO - Biochemistry
JF - Biochemistry
SN - 0006-2960
IS - 37
ER -