Radke G A, Ono K, Ravindran S, Roche T E
Department of Biochemistry, Kansas State University, Manhattan 66506.
Biochem Biophys Res Commun. 1993 Feb 15;190(3):982-91. doi: 10.1006/bbrc.1993.1146.
Mammalian pyruvate dehydrogenase kinase binds to the lipoyl domain region of the core structure forming dihydrolipoyl acetyltransferase (E2) subunits. The bound kinase has a greatly enhanced rate in phosphorylating E2-bound pyruvate dehydrogenase (E1) tetramers versus the rate at which resolved kinase phosphorylates dissociated E1. This E2-activated kinase function was completely prevented by selective alkylation of reduced lipoyl groups while kinase and E1 binding to the E2 core were retained. Selective removal of lipoyl cofactors from intact E2 by treatment with Enterococcus faecalis lipoamidase decreased kinase activity by 4-fold and caused selective release of a major portion of the kinase from E2 in a sucrose-step gradient procedure. Selective and reversible modification of the lipoyl groups of E2 subunits also allowed the kinase to be dissociated under mildly chaotropic conditions. Thus, the lipoyl prosthetic group on one of the two lipoyl domains of E2 subunits is critically important for maintaining E2-activated kinase function and contributes to binding of the kinase to E2. Since removal of the lipoyl group weakened kinase binding to E2 more than modifying lipoyl thiols, it is suggested that the hydrophobic inner portion of the lipoyl conjugate (i.e., lysine carbons and C1 to C5 of the lipoic acid) is important in the binding of the kinase.
哺乳动物丙酮酸脱氢酶激酶与核心结构的二氢硫辛酰乙酰转移酶（E2）亚基的硫辛酰结构域区域结合。与游离激酶磷酸化解离的E1的速率相比，结合的激酶对E2结合的丙酮酸脱氢酶（E1）四聚体进行磷酸化的速率大大提高。通过对还原的硫辛酰基团进行选择性烷基化，可完全阻止这种E2激活的激酶功能，同时保留激酶与E1与E2核心的结合。用粪肠球菌硫辛酰胺酶处理完整的E2，选择性去除硫辛酰辅因子，可使激酶活性降低4倍，并在蔗糖阶梯梯度实验中导致大部分激酶从E2中选择性释放。E2亚基硫辛酰基团的选择性和可逆修饰也使激酶在轻度离液条件下解离。因此，E2亚基两个硫辛酰结构域之一上的硫辛酰辅基对于维持E2激活的激酶功能至关重要，并有助于激酶与E2的结合。由于去除硫辛酰基团比修饰硫辛酰硫醇更能削弱激酶与E2的结合，因此表明硫辛酰共轭物的疏水内部（即硫辛酸的赖氨酸碳和C1至C5）在激酶的结合中很重要。
