Balme A, Lederer F
CNRS URA 1461, Hôpital Necker, Paris, France.
Protein Sci. 1994 Jan;3(1):109-17. doi: 10.1002/pro.5560030114.
The family of FMN-dependent, alpha-hydroxy acid-oxidizing enzymes catalyzes substrate dehydrogenation by a mechanism the first step of which is abstraction of the substrate alpha-proton (so-called carbanion mechanism). For flavocytochrome b2 and lactate oxidase, it was shown that once on the enzyme this proton is lost only slowly to the solvent (Lederer F, 1984, In: Bray RC, Engel PC, Mayhew SG, eds, Flavins & flavoproteins, Berlin: Walter de Gruyter & Co., pp 513-526; Urban P, Lederer F, 1985, J Biol Chem 260:11115-11122). This suggested the occurrence of a pKa increase of the catalytic histidine upon enzyme reduction by substrate. For flavocytochrome b2, the crystal structure indicated 2 possible origins for the stabilization of the imidazolium form of His 373: either a network of hydrogen bonds involving His 373, Tyr 254, flavin N5 and O4, a heme propionate, and solvent molecules, and/or electrostatic interactions with Asp 282 and with the reduced cofactor N1 anion. In this work, we probe the effect of the hydrogen bond network at the active site by studying proton exchange with solvent for 2 mutants: Y254F and the recombinant flavodehydrogenase domain, in which this network should be disrupted. The rate of proton exchange, as determined by intermolecular hydrogen transfer experiments, appears identical in the flavodehydrogenase domain and the wild-type enzyme, whereas it is about 3-fold faster in the Y254F mutant. It thus appears that specific hydrogen bonds to the solvent do not play a major role in stabilizing the acid form of His 373 in reduced flavocytochrome b2. Removal of the Y254 phenol group induces a pKa drop of about half a pH unit for His 373 in the reduced enzyme. Even then, the rate of exchange of the imidazolium proton with solvent is still lower by several orders of magnitude than that of a normally ionizing histidine. Other factors must then also contribute to the pKa increase, such as the electrostatic interactions with D282 and the anionic reduced cofactor, as suggested by the crystal structure.
依赖黄素单核苷酸（FMN）的α-羟酸氧化酶家族通过一种机制催化底物脱氢，该机制的第一步是夺取底物的α-质子（所谓的碳负离子机制）。对于黄素细胞色素b2和乳酸氧化酶，研究表明，一旦质子与酶结合，它只会缓慢地释放到溶剂中（Lederer F，1984年，见：Bray RC、Engel PC、Mayhew SG编，《黄素与黄素蛋白》，柏林：Walter de Gruyter & Co.，第513 - 526页；Urban P、Lederer F，1985年，《生物化学杂志》260:11115 - 11122）。这表明在底物使酶还原后，催化性组氨酸的pKa会升高。对于黄素细胞色素b2，晶体结构显示了His 373咪唑鎓形式稳定化的2个可能来源：要么是涉及His 373、Tyr 254、黄素N5和O4、一个血红素丙酸酯以及溶剂分子的氢键网络，和/或与Asp 282以及还原辅因子N1阴离子的静电相互作用。在这项工作中，我们通过研究2个突变体与溶剂的质子交换来探究活性位点氢键网络的作用：Y254F和重组黄素脱氢酶结构域，在其中这个网络应该被破坏。通过分子间氢转移实验测定的质子交换速率，在黄素脱氢酶结构域和野生型酶中似乎是相同的，而在Y254F突变体中大约快3倍。因此，与溶剂的特定氢键在稳定还原型黄素细胞色素b2中His 373的酸形式方面似乎并不起主要作用。去除Y254酚基会使还原型酶中His 373的pKa下降约半个pH单位。即便如此，咪唑鎓质子与溶剂的交换速率仍然比正常电离的组氨酸低几个数量级。那么其他因素也必定对pKa升高有贡献，如晶体结构所表明的，与D282以及阴离子还原辅因子的静电相互作用。
