Program in Biochemistry and Molecular Biology (BMB), Boston University , Boston, Massachusetts 02215, United States.
Department of Physiology and Biophysics, Boston University School of Medicine , Boston, Massachusetts 02118, United States .
J Am Chem Soc. 2015 Nov 4;137(43):13876-86. doi: 10.1021/jacs.5b08149. Epub 2015 Oct 21.
Substrate recognition is one of the hallmarks of enzyme catalysis. Enzyme conformational changes have been linked to selectivity between substrates with little direct evidence. Aldolase, a glycolytic enzyme, must distinguish between two physiologically important substrates, fructose 1-phosphate and fructose 1,6-bisphosphate, and provides an excellent model system for the study of this question. Previous work has shown that isozyme specific residues (ISRs) distant from the active site are responsible for kinetic distinction between these substrates. Notably, most of the ISRs reside in a cluster of five surface α-helices, and the carboxyl-terminal region (CTR), and cooperative interactions among these helices have been demonstrated. To test the hypothesis that conformational changes are at the root of these changes, single surface-cysteine variants were created with the cysteine located on helices of the cluster and CTR. This allowed for site-specific labeling with an environmentally sensitive fluorophore, and subsequent monitoring of conformational changes by fluorescence emission spectrophotometry. These labeled variants revealed different spectra in the presence of saturating amounts of each substrate, which suggested the occurrence of different conformations. Emission spectra collected at various substrate concentrations showed a concentration dependence of the fluorescence spectra, consistent with binding events. Lastly, stopped-flow fluorescence spectrophotometry showed that the rate of these fluorescence changes was on the same time-scale as catalysis, thus suggesting a link between the different fluorescence changes and events during catalysis. On the basis of these results, we propose that different conformational changes may be a common mechanism for dictating substrate specificity in other enzymes with multiple substrates.
底物识别是酶催化的特点之一。虽然酶构象变化与底物选择性之间存在关联，但缺乏直接证据。醛缩酶是一种糖酵解酶，必须区分两种生理上重要的底物，即果糖 1-磷酸和果糖 1,6-二磷酸，它为研究这一问题提供了一个极好的模型系统。先前的工作表明，同工酶特异性残基（ISRs）远离活性位点，负责这些底物之间的动力学区分。值得注意的是，大多数 ISRs 位于五个表面α-螺旋簇和羧基末端区域（CTR）中，并且已经证明了这些螺旋之间的协同相互作用。为了测试构象变化是这些变化根源的假设，创建了具有位于簇和 CTR 中螺旋上的半胱氨酸的单个表面半胱氨酸变体。这允许使用环境敏感荧光团进行位点特异性标记，并通过荧光发射分光光度法随后监测构象变化。这些标记的变体在存在每种底物的饱和量时显示出不同的光谱，这表明发生了不同的构象。在各种底物浓度下收集的发射光谱显示荧光光谱与浓度有关，这与结合事件一致。最后，停流荧光分光光度法表明，这些荧光变化的速率与催化速率相同，因此表明在催化过程中不同的荧光变化与事件之间存在联系。基于这些结果，我们提出不同的构象变化可能是其他具有多种底物的酶决定底物特异性的共同机制。
