Schnackerz K D, Mozzarelli A
Theodor-Boveri-Institut für Biowissenschaften, Physiologische Chemie I, Am Hubland, D-97074 Würzburg, Germany.
J Biol Chem. 1998 Dec 11;273(50):33247-53. doi: 10.1074/jbc.273.50.33247.
The functional properties of tryptophan synthase alpha2beta2 complex are modulated by a variety of allosteric effectors, including pH, monovalent cations, and alpha-subunit ligands. The dynamic properties of the beta-active site were probed by 31P NMR spectroscopy of the enzyme-bound coenzyme pyridoxal 5'-phosphate. The 31P NMR signal of the cofactor phosphate of the internal aldimine exhibits a single peak at 3.73 ppm with a line width of 12 Hz. In the presence of saturating concentrations of sodium ions, the 31P signal shifts to 3.97 ppm concomitant with a change in line width to 35 Hz. The latter indicates that sodium ions decrease the conformational flexibility of the coenzyme. In the absence of ions, lowering pH leads to the appearance of a second peak at 4.11 ppm, the intensity of which decreases in the presence of cesium ions. Addition of L-serine in the presence of sodium ions leads to the formation of the external aldimine, the first metastable catalytic intermediate. The 31P signal does not change its position, but a change in line width from 35 to 5 Hz is observed, revealing that this species is characterized by a considerable degree of rotational freedom around the coenzyme C-O bond. In the presence of L-serine and either cesium ions or the allosteric effector indole-3-acetylglycine, the accumulation of the second catalytic intermediate, alpha-aminoacrylate, is observed. The 31P signal is centered at 3.73 ppm with a line width of 5 Hz, indicating that the phosphate group of the coenzyme in the external aldimine and the alpha-aminoacrylate exhibits the same flexibility but a slightly different state of ionization. Because the alpha-aminoacrylate intermediate but not the external aldimine triggers the allosteric signal to the alpha-subunit, other portions of the beta-active site modify their dynamic properties in response to the progress of the catalytic process. A narrow line width was also observed for the quinonoid species formed by nucleophilic attack of indoline to the alpha-aminoacrylate. The 31P signal moves downfield to 4.2 ppm, indicating a possible change of the ionization state of the phosphate group. Thus, the modification of either the ionization state of the coenzyme phosphate or its flexibility or both are, at least in part, responsible for the conformational events that accompany the catalytic process.
色氨酸合成酶α2β2复合物的功能特性受到多种别构效应剂的调节，包括pH、单价阳离子和α亚基配体。通过对与酶结合的辅酶磷酸吡哆醛进行31P NMR光谱研究，探究了β活性位点的动态特性。内部醛亚胺的辅因子磷酸基团的31P NMR信号在3.73 ppm处呈现单峰，线宽为12 Hz。在存在饱和浓度钠离子的情况下，31P信号移至3.97 ppm，同时线宽变为35 Hz。后者表明钠离子降低了辅酶的构象灵活性。在没有离子的情况下，降低pH会导致在4.11 ppm处出现第二个峰，在铯离子存在时其强度会降低。在钠离子存在的情况下添加L-丝氨酸会导致形成外部醛亚胺，即第一个亚稳态催化中间体。31P信号位置不变，但观察到线宽从35 Hz变为5 Hz，这表明该物种的特征是围绕辅酶C-O键具有相当程度的旋转自由度。在存在L-丝氨酸和铯离子或别构效应剂吲哚-3-乙酰甘氨酸的情况下，会观察到第二个催化中间体α-氨基丙烯酸酯的积累。31P信号集中在3.73 ppm，线宽为5 Hz，这表明外部醛亚胺和α-氨基丙烯酸酯中辅酶的磷酸基团具有相同的灵活性，但电离状态略有不同。由于α-氨基丙烯酸酯中间体而非外部醛亚胺触发了向α亚基的别构信号，β活性位点的其他部分会根据催化过程的进展改变其动态特性。对于由二氢吲哚对α-氨基丙烯酸酯进行亲核攻击形成的醌型物种，也观察到了窄线宽。31P信号向低场移动至4.2 ppm，表明磷酸基团的电离状态可能发生了变化。因此，辅酶磷酸基团的电离状态或其灵活性或两者的改变至少部分地导致了伴随催化过程的构象变化。
