Rapid loop dynamics of Yersinia protein tyrosine phosphatases
Rapid loop dynamics of Yersinia protein tyrosine phosphatases
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DOI：
10.1021/bi9622130
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发表时间：
1997-02-25
期刊：
影响因子：
2.9
通讯作者：
Eads, DD
中科院分区：
文献类型：
--
作者：
Juszczak, LJ;Zhang, ZY;Eads, DD
耶尔森氏菌蛋白酪氨酸磷酸酶 (PTPase) 含有一个单一且不变的色氨酸 (W354)，位于柔性环（WpD 环）的铰链位置之一，这对于催化至关重要。使用时间分辨荧光各向异性和稳态紫外共振拉曼 (UVRR) 光谱检查了野生型耶尔森氏菌 PTP 酶和活性位点突变体，其中必需的 Cys 403 已被丝氨酸 (C403S) 取代。在有和没有结合抑制剂砷酸盐的情况下检查这两种酶。 UVRR 光谱表明，在溶液中，无配体的野生型 PTPase 作为两个色氨酸旋转异构体结构的平衡混合物存在，chi(2,1) 二面角为 -4 度和 -90 度。这两种旋转异构体归因于无配体酶的“闭合”和“开放”WpD 环构象异构体的存在。相反，砷酸盐连接的野生型 PTPase 和无配体且砷酸盐连接的 C403S PTPase 的 UVRR 光谱包含与 W354 的 -4 度旋转异构体相关的单个 W3 带，表明闭合 WpD 环构象异构体占主导地位。配体结合的野生型耶尔森氏菌 PTPase 和 C403S PTPase 的两种连接状态的色氨酸荧光各向异性衰减测量显示，由于蛋白质的旋转运动，单个相关时间为 30-48 ns，而无配体的野生型 PTPase 被发现具有 31 和 3.8 ns 的两个相关时间。无配体酶的 3.8 ns 相关时间归因于包含 W354 的 WpD 环的铰链运动。这些结果表明，在生理条件下，未连接的野生型耶尔森氏菌 PTPase 在开放 WpD 环和闭环形式之间交替，速率常数类似于 2.6 x 10(8) s(-1)。我们得出结论，野生型耶尔森氏菌 PTPase 的 WpD 环闭合速率因此与配体的存在无关，而在配体存在下，打开速率显着降低，导致配体结合的闭合构象。相比之下，无配体和连接的 C403S PTPase 在我们的动态测量过程中保持环闭合构型。 C403S PTPase 中缺乏 WpD 环运动被认为是由于 WpD 环的阴离子硫醇盐和 Asp 356 之间排斥势的损失和/或 Ser 403 和 Asp 356 之间形成氢键或水桥氢键。
The Yersinia protein tyrosine phosphatases (PTPase) contain a single and invariant tryptophan (W354) located at one of the hinge positions of the flexible loop (WpD loop), which is essential for catalysis. The wild-type Yersinia PTPase and an active site mutant in which the esential Cys 403 has been replaced by serine (C403S) have been examined using both time-resolved fluorescence anisotropy and steady-state UV resonance Raman (UVRR) spectroscopies. Both enzymes were examined with and without the bound inhibitor arsenate. The UVRR spectra indicate that in solution the ligand-free, wildtype PTPase exists as an equilibrium mixture of two tryptophan rotamer structures with chi(2,1) dihedral angles of -4 degrees and -90 degrees. The two rotamers have been attributed to the presence of both ''closed'' and ''open'' WpD loop conformers of the ligand-free enzyme. Conversely, the UVRR spectra of the arsenate-ligated, wild-type PTPase and of ligand-free and arsenate-ligated C403S PTPase contain a single W3 band which is correlated to the -4 degrees rotamer of W354, indicating a predominance of the closed WpD loop conformer. The tryptophan fluorescence anisotropy decay measurements of the ligand-bound, wild-type Yersinia PTPase and of both ligation states of the C403S PTPase reveal a single correlation time of 30-48 ns due to the rotational motion of the protein, while the ligand-free, wild-type PTPase is found to have two correlation times of 31 and 3.8 ns. The 3.8 ns correlation time of the ligand-free enzyme is attributed to the hinged movement of the WpD loop which contains W354. These results indicate that under physiological conditions, the nonligated, wild-type Yersinia PTPase alternates between an open WpD loop and a closed loop form with a rate constant of similar to 2.6 x 10(8) s(-1). We conclude that the rate of WpD loop closure of the wild-type Yersinia PTPase is thus independent of the presence of ligand, whereas in the presence of ligand the rate of opening is dramatically reduced resulting in a closed conformation on ligand binding. In contrast, the ligand-free and ligated C403S PTPase remain in the loop closed configuration over the time course of our dynamic measurements. The lack of WpD loop motion in the C403S PTPase is believed to be due to either a loss of repulsive potential between the anionic thiolate and Asp 356 of the WpD loop and/or the formation of a hydrogen bond or water bridged hydrogen bond between Ser 403 and Asp 356.