Olsen K, Christensen U, Sierks M R, Svensson B
Chemical Laboratory IV, University of Copenhagen, Denmark.
Biochemistry. 1993 Sep 21;32(37):9686-93. doi: 10.1021/bi00088a021.
Interactions of wild-type and Trp120-->Phe glucoamylase with maltooligodextrin (Gx) 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 + Gx(or A) (k1) <==> (k-1) EGx (or A) (k2) <==> (k-2) E*Gx(or A) (k3) --> E + P or E-A, previously shown to account for the glucoamylase-maltose system [Olsen, K., Svensson, B., & 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 kc 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.
本文采用停流荧光光谱法和稳态动力学测量法，研究了野生型和色氨酸120突变为苯丙氨酸的葡萄糖淀粉酶与麦芽糊精（Gx）底物以及紧密结合抑制剂阿卡波糖（A）之间的相互作用。所有野生型和色氨酸120突变为苯丙氨酸的葡萄糖淀粉酶反应均遵循三步模型：E + Gx（或A）（k1）⇌（k - 1）EGx（或A）（k2）⇌（k - 2）E*Gx（或A）（k3）→E + P或E - A，该模型先前已被证明适用于葡萄糖淀粉酶 - 麦芽糖系统[奥尔森，K.，斯文森，B.，& 克里斯蒂安森，U.（1992年）《欧洲生物化学杂志》209卷，777 - 784页]。确定了K1 = k - 1/k1、k2、k - 2以及催化常数k3。在第一步反应中，麦芽糊精的结合较弱，野生型和色氨酸120突变为苯丙氨酸的葡萄糖淀粉酶之间差异不大。相比之下，涉及构象变化的第二步受到突变和底物长度的强烈影响。在这里，野生型葡萄糖淀粉酶反应更快，底物越长形成的中间体越稳定。相反，色氨酸120突变为苯丙氨酸的葡萄糖淀粉酶底物越长反应越慢。因此，突变对麦芽糖的影响最小。色氨酸120突变为苯丙氨酸仅使荧光信号降低12 - 20%，这表明其他色氨酸残基在报告构象变化中很重要。色氨酸120也强烈影响实际的催化步骤，因为该突变使kc值降低了30 - 80倍。在第一步和第二步中，阿卡波糖与野生型葡萄糖淀粉酶的相互作用类似于麦芽四糖，但与色氨酸120突变为苯丙氨酸的葡萄糖淀粉酶不同。此外，阿卡波糖反应中与催化步骤平行的第三步受到突变的强烈影响。速率常数k3增加了200倍。
