Grant G A, Xu X L
Department of Molecular Biology and Pharmacology and the Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1998 Aug 28;273(35):22389-94. doi: 10.1074/jbc.273.35.22389.
D-3-Phosphoglycerate dehydrogenase from Escherichia coli is a homotetrameric enzyme which is allosterically regulated by the end product of its pathway, L-serine. The enzyme binds 4 L-serine molecules at two interfaces formed by the noncovalent association of the regulatory domains. The two domains that comprise each interface are related by an approximately 180 degrees axis of symmetry, and two serine molecules bind at each interface by forming a hydrogen bond network between the domains. A model has been proposed that suggests that serine functions by drawing adjacent domains together and that this in turn translates a conformational change to the active site. A tryptophan residue has been engineered into the helices flanking the regulatory interfaces that displays significant quenching in response to serine binding. Residues on the adjacent subunit appear to be primarily responsible for the tryptophan quenching and thus support the hypothesis that serine binding leads to an increase in the proximity between residues on neighboring subunits. Serine binding studies show that this quenching, as well as inhibition of enzymatic activity, are essentially complete when only two of the four serine binding sites are occupied. The requirement for only one serine per interface is consistent with the notion that the interface is formed by relatively rigid domains and that hydrogen bonding at only a single site is all that is required to substantially close the interface. The fluorescence quenching in response to L-serine binding generally correlates with enzymatic inhibition, but there appears to be a slight lag in inhibition relative to quenching at low serine concentrations. The observed fluorescence quenching of residues in the regulatory domains of D-3-phosphoglycerate dehydrogenase provide the first direct evidence for a conformational change in response to effector binding and provide a means to monitor the first step in the allosteric mechanism.
来自大肠杆菌的D-3-磷酸甘油酸脱氢酶是一种同四聚体酶，受其代谢途径终产物L-丝氨酸的变构调节。该酶在由调节结构域非共价结合形成的两个界面处结合4个L-丝氨酸分子。构成每个界面的两个结构域通过大约180度的对称轴相关联，并且两个丝氨酸分子通过在结构域之间形成氢键网络而在每个界面处结合。有人提出了一个模型，表明丝氨酸通过将相邻结构域拉到一起发挥作用，进而将构象变化传递到活性位点。一个色氨酸残基已被设计到调节界面两侧的螺旋中，该色氨酸残基在结合丝氨酸时表现出显著的淬灭。相邻亚基上的残基似乎是色氨酸淬灭的主要原因，因此支持了丝氨酸结合导致相邻亚基上残基之间距离增加的假设。丝氨酸结合研究表明，当四个丝氨酸结合位点中只有两个被占据时，这种淬灭以及酶活性的抑制基本完成。每个界面只需要一个丝氨酸与以下观点一致，即界面由相对刚性的结构域形成，并且仅在单个位点的氢键结合就足以基本封闭界面。响应L-丝氨酸结合的荧光淬灭通常与酶抑制相关，但在低丝氨酸浓度下，抑制相对于淬灭似乎有轻微延迟。观察到的D-3-磷酸甘油酸脱氢酶调节结构域中残基的荧光淬灭为效应物结合引起的构象变化提供了首个直接证据，并提供了一种监测变构机制第一步的方法。
