Kato Masato, Wynn R Max, Chuang Jacinta L, Tso Shih-Chia, Machius Mischa, Li Jun, Chuang David T
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75390-9038, USA.
Structure. 2008 Dec 10;16(12):1849-59. doi: 10.1016/j.str.2008.10.010.
We report the crystal structures of the phosporylated pyruvate dehydrogenase (E1p) component of the human pyruvate dehydrogenase complex (PDC). The complete phosphorylation at Ser264-alpha (site 1) of a variant E1p protein was achieved using robust pyruvate dehydrogenase kinase 4 free of the PDC core. We show that unlike its unmodified counterpart, the presence of a phosphoryl group at Ser264-alpha prevents the cofactor thiamine diphosphate-induced ordering of the two loops carrying the three phosphorylation sites. The disordering of these phosphorylation loops is caused by a previously unrecognized steric clash between the phosphoryl group at site 1 and a nearby Ser266-alpha, which nullifies a hydrogen-bonding network essential for maintaining the loop conformations. The disordered phosphorylation loops impede the binding of lipoyl domains of the PDC core to E1p, negating the reductive acetylation step. This results in the disruption of the substrate channeling in the PDC, leading to the inactivation of this catalytic machine.
我们报道了人类丙酮酸脱氢酶复合体（PDC）的磷酸化丙酮酸脱氢酶（E1p）组分的晶体结构。使用不含PDC核心的活性丙酮酸脱氢酶激酶4实现了变体E1p蛋白Ser264-α（位点1）的完全磷酸化。我们发现，与未修饰的对应物不同，Ser264-α处存在磷酸基团会阻止辅因子硫胺二磷酸诱导的携带三个磷酸化位点的两个环的有序化。这些磷酸化环的无序化是由位点1处的磷酸基团与附近的Ser266-α之间先前未被认识的空间冲突引起的，这使维持环构象所必需的氢键网络无效。无序的磷酸化环阻碍了PDC核心的硫辛酰结构域与E1p的结合，使还原乙酰化步骤无效。这导致了PDC中底物通道的破坏，导致这种催化机制失活。
