Juszczak L J, Zhang Z Y, Wu L, Gottfried D S, Eads D D
Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461, USA.
Biochemistry. 1997 Feb 25;36(8):2227-36. doi: 10.1021/bi9622130.
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 The wild-type Yersinia PTPase and an active site mutant in which the esential Cys 403 has been replaced by serine (C403S) have been examined using both time-resolved fluorescence anisotropy and steady-state UV resonance Raman (UVRR) spectroscopies.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, wild-type PTPase exists as an equilibrium mixture of two tryptophan rotamer structures with chi2,1 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 approximately 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.
耶尔森氏菌蛋白酪氨酸磷酸酶（PTPase）在柔性环（WpD环）的一个铰链位置含有一个单一且不变的色氨酸（W354），这对催化作用至关重要。已使用时间分辨荧光各向异性和稳态紫外共振拉曼（UVRR）光谱对野生型耶尔森氏菌PTPase以及活性位点突变体（其中必需的半胱氨酸403已被丝氨酸取代，即C403S）进行了研究。两种酶在有和没有结合抑制剂砷酸盐的情况下都进行了检测。UVRR光谱表明，在溶液中，无配体的野生型PTPase以两种色氨酸旋转异构体结构的平衡混合物形式存在，其二面角chi2,1分别为-4度和-90度。这两种旋转异构体被认为分别对应无配体酶的“闭合”和“开放”WpD环构象。相反，砷酸盐结合的野生型PTPase以及无配体和砷酸盐结合的C403S PTPase的UVRR光谱都只包含一个W3带，该带与W354的-4度旋转异构体相关，表明闭合的WpD环构象占主导。对结合配体的野生型耶尔森氏菌PTPase以及C403S PTPase两种结合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，单一相关时间为30 - 48纳秒，而无配体的野生型PTPase有31纳秒和3.8纳秒两个相关时间。无配体酶的3.8纳秒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，未结合配体的野生型耶尔森氏菌PTPase以大约2.6×10(8) s(-1)的速率常数在开放的WpD环和闭合环形式之间交替。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，而在有配体存在时，开放速率显著降低，导致在配体结合时形成闭合构象。相比之下，在我们的动态测量过程中，无配体和结合配体的C403S PTPase都保持环闭合构型。据信，C403S PTPase中WpD环缺乏运动是由于阴离子硫醇盐与WpD环的天冬氨酸356之间排斥电位的丧失和/或丝氨酸403与天冬氨酸356之间形成氢键或水桥连氢键所致。
