Karmodiya Krishanpal, Surolia Namita
Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Jakkur, Bangalore 560064, India.
Proteins. 2008 Feb 1;70(2):528-38. doi: 10.1002/prot.21530.
The urea and guanidinium chloride (GdmCl) induced unfolding of FabG, a beta-ketoacyl-ACP reductase of Plasmodium falciparum, was examined in detail using intrinsic fluorescence of FabG, UV-circular dichroism (CD), spectrophotometric enzyme activity measurements, glutaraldehyde cross-linking, and size exclusion chromatography. The equilibrium unfolding of FabG by urea is a multistep process as compared with a two-state process by GdmCl. FabG is fully unfolded at 6.0M urea and 4.0M GdmCl. Approximately 90% of the enzyme activity could be recovered on dialyzing the denaturants, showing that denaturation by both urea and GdmCl is reversible. We found two states in the reversible unfolding process of FabG in presence of NADPH; one is an activity-enhanced state and the other, an inactive state in case of equilibrium unfolding with urea. On the contrary, in presence of NADPH, there is no stabilization of FabG in case of equilibrium unfolding with GdmCl. We hypothesize that the hydrogen-bonding network may be reorganized by the denaturant in the activity-enhanced state formed in presence of 1.0M urea, by interrupting the association between dimer-dimer interface and help in accommodating the larger substrate in the substrate binding tunnel thus, increasing the activity. Furthermore, binding of the active site organizer, NADPH leads to compaction of the FabG in presence of urea, as evident by acrylamide quenching. We have shown here for the first time, the detailed inactivation kinetics of FabG, which have not been evaluated in the past from any of the FabG family of enzymes from any of the other sources. These findings provide impetus for exploring the influences of ligands on the structure-activity relationship of Plasmodium beta-ketoacyl-ACP reductase.
利用法布G（FabG）的内在荧光、紫外圆二色性（CD）、分光光度法酶活性测量、戊二醛交联和尺寸排阻色谱法，详细研究了尿素和氯化胍（GdmCl）诱导的恶性疟原虫β-酮酰基-ACP还原酶法布G的去折叠过程。与GdmCl诱导的两态过程相比，尿素诱导的法布G平衡去折叠是一个多步过程。法布G在6.0M尿素和4.0M GdmCl中完全去折叠。透析变性剂后，约90%的酶活性可以恢复，这表明尿素和GdmCl诱导的变性都是可逆的。我们发现在存在NADPH的情况下，法布G的可逆去折叠过程中有两个状态；一个是活性增强状态，另一个是在与尿素平衡去折叠时的无活性状态。相反，在存在NADPH的情况下，与GdmCl平衡去折叠时法布G没有稳定化。我们假设，在1.0M尿素存在下形成的活性增强状态中，变性剂可能会通过中断二聚体-二聚体界面之间的缔合来重组氢键网络，并有助于在底物结合通道中容纳更大的底物，从而提高活性。此外，活性位点组织者NADPH的结合导致在存在尿素的情况下法布G发生压缩，这通过丙烯酰胺猝灭得以证明。我们首次在此展示了法布G的详细失活动力学，过去从未对来自任何其他来源的法布G酶家族进行过评估。这些发现为探索配体对疟原虫β-酮酰基-ACP还原酶结构-活性关系的影响提供了动力。
