Majamaa K, Günzler V, Hanauske-Abel H M, Myllylä R, Kivirikko K I
J Biol Chem. 1986 Jun 15;261(17):7819-23.
Various hydroxybenzenes, hydroxybenzoic acids, and related compounds resemble structurally both 2-oxoglutarate and ascorbate, two reactants needed in the reaction of prolyl 4-hydroxylase. These substances were found to inhibit prolyl 4-hydroxylase competitively with respect to both cosubstrates. Ortho-dihydroxy derivatives, which are capable of chelating the enzyme-bound iron, were the most effective inhibitors, with Ki values of about 5 microM. In contrast, pyridine 2-carboxylates, which have previously been reported to inhibit the enzyme competitively with respect to 2-oxoglutarate, were found to inhibit it uncompetitively with respect to ascorbate. In a separate set of experiments the side chain of the ascorbate molecule was shown to make no significant contribution to the binding of the reductant to the enzyme, as D(-)-isoascorbate and 5,6-O-isopropylidene ascorbate gave essentially the same Vmax and Km values as ascorbate. On the other hand, structural modifications of the ring atoms that abolished the chelating capacity destroyed both the cosubstrate and inhibitory activity, as in L-galactono gamma-lactone. The ascorbate binding site therefore appears to consist of two cis-positioned coordination sites of the enzyme-bound iron and is thus partially identical to the binding site of 2-oxoglutarate. This mode of interaction suggests that ascorbate reduces the enzyme-bound iron through an "inner-sphere" mechanism. The inhibitors studied appear to react at different phases of the catalytic cycle, determined by the oxidation state of the enzyme-bound iron atom.
多种羟基苯、羟基苯甲酸及相关化合物在结构上既类似于2-氧代戊二酸，又类似于抗坏血酸，这两种物质是脯氨酰4-羟化酶反应所需的反应物。已发现这些物质对两种共底物而言均竞争性抑制脯氨酰4-羟化酶。能够螯合酶结合铁的邻二羟基衍生物是最有效的抑制剂，其Ki值约为5微摩尔。相比之下，先前报道的吡啶2-羧酸盐对2-氧代戊二酸而言竞争性抑制该酶，但对抗坏血酸而言非竞争性抑制该酶。在另一组实验中，抗坏血酸分子的侧链对还原剂与酶的结合无显著贡献，因为D(-)-异抗坏血酸和5,6-O-异亚丙基抗坏血酸给出的Vmax和Km值与抗坏血酸基本相同。另一方面，如L-半乳糖酸γ-内酯那样，消除螯合能力的环原子结构修饰破坏了共底物和抑制活性。因此，抗坏血酸结合位点似乎由酶结合铁的两个顺式配位位点组成，因此部分与2-氧代戊二酸的结合位点相同。这种相互作用模式表明抗坏血酸通过“内球”机制还原酶结合铁。所研究的抑制剂似乎在催化循环的不同阶段发生反应，这由酶结合铁原子的氧化态决定。
