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)|
|Number of pages||8|
|Publication status||Published - 1993|
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