Grant G A, Schuller D J, Banaszak L J
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Protein Sci. 1996 Jan;5(1):34-41. doi: 10.1002/pro.5560050105.
Escherichia coli D-3-phosphoglycerate dehydrogenase (ePGDH) is a tetramer of identical subunits that is allosterically inhibited by L-serine, the end product of its metabolic pathway. Because serine binding affects the velocity of the reaction and not the binding of substrate or cofactor, the enzyme is classified as of the Vmax type. Inhibition by a variety of amino acids and analogues of L-serine indicate that all three functional groups of serine are required for optimal interaction. Removing or altering any one functional group results in an increase in inhibitory concentration from micromolar to millimolar, and removal or alteration of any two functional groups removes all inhibitory ability. Kinetic studies indicate at least two serine-binding sites, but the crystal structure solved in the presence of bound serine and direct serine-binding studies show that there are a total of four serine-binding sites on the enzyme. However, approximately 85% inhibition is attained when only two sites are occupied. The three-dimensional structure of ePGDH shows that the serine-binding sites reside at the interface between regulatory domains of adjacent subunits. Two serine molecules bind at each of the two regulatory domain interfaces in the enzyme. When all four of the serines are bound, 100% inhibition of activity is seen. However, because the domain contacts are symmetrical, the binding of only one serine at each interface is sufficient to produce approximately 85% inhibition. The tethering of the regulatory domains to each other through multiple hydrogen bonds from serine to each subunit apparently prevents the body of these domains from undergoing the reorientation that must accompany a catalytic cycle. It is suggested that part of the conformational change may involve a hinge formed in the vicinity of the union of two antiparallel beta-sheets in the regulatory domains. The tethering function of serine, in turn, appears to prevent the substrate-binding domain from closing the cleft between it and the nucleotide-binding domain, which may be necessary to form a productive hydrophobic environment for hydride transfer. Thus, the structure provides a plausible model that is consistent with the binding and inhibition data and that suggests that catalysis and inhibition in this rare Vmax-type allosteric enzyme is based on the movement of rigid domains about flexible hinges.
大肠杆菌D-3-磷酸甘油酸脱氢酶（ePGDH）是由相同亚基组成的四聚体，其代谢途径的终产物L-丝氨酸可对其进行变构抑制。由于丝氨酸结合影响反应速度，而非底物或辅因子的结合，因此该酶被归类为Vmax型。多种氨基酸和L-丝氨酸类似物的抑制作用表明，丝氨酸的所有三个官能团对于最佳相互作用都是必需的。去除或改变任何一个官能团都会导致抑制浓度从微摩尔增加到毫摩尔，而去除或改变任何两个官能团则会消除所有抑制能力。动力学研究表明至少有两个丝氨酸结合位点，但在结合丝氨酸存在下解析的晶体结构以及直接的丝氨酸结合研究表明，该酶上共有四个丝氨酸结合位点。然而，当只有两个位点被占据时，可达到约85%的抑制率。ePGDH的三维结构表明，丝氨酸结合位点位于相邻亚基调节结构域之间的界面处。两个丝氨酸分子结合在酶的两个调节结构域界面的每一个上。当所有四个丝氨酸都结合时，可观察到100%的活性抑制。然而，由于结构域接触是对称的，每个界面仅结合一个丝氨酸就足以产生约85%的抑制。调节结构域通过丝氨酸与每个亚基之间的多个氢键相互连接，这显然阻止了这些结构域的主体发生伴随催化循环必须经历的重新定向。有人提出，构象变化的一部分可能涉及在调节结构域中两个反平行β-折叠结合处附近形成的铰链。丝氨酸的连接功能反过来似乎阻止了底物结合结构域关闭其与核苷酸结合结构域之间的裂隙，这对于形成用于氢化物转移的有效疏水环境可能是必要的。因此，该结构提供了一个合理的模型，与结合和抑制数据一致，并表明这种罕见的Vmax型变构酶中的催化和抑制作用基于刚性结构域围绕柔性铰链的移动。
