Yamashita A, Nagatsuka T, Watanabe M, Kondo H, Sugiura T, Waku K
Faculty of Pharmaceutical Sciences, Teikyo University, Kanagawa, Japan.
Biochem Pharmacol. 1997 Feb 21;53(4):561-70. doi: 10.1016/s0006-2952(96)00793-9.
We previously identified and purified UDP-glucuronosyltransferase (UGT) isoforms as targets of protein acylation from rat liver microsomes (Yamashita et al., Biochem J 312: 301-308, 1995). The acylation of UGT isoforms occurred upon incubation with acyl-CoA without another protein acyltransferase, suggesting that it was autoacylation. The study revealed the interaction of UGT isoforms with acyl-CoA. In the present study, the effects of fatty acyl-CoA on UGT activities were examined thoroughly, using a rat liver microsomal and purified enzyme fractions. The UGT activities of both fractions were inhibited by acyl-CoA in a concentration-dependent manner. The effect of acyl-CoA was observed on the activities toward various substrates, suggesting that the effect shows the wide spectrum of the isoforms of UGT. To assess the mechanism underlying the inhibition of UGT activity by acyl-CoA, the relationship of the inhibition, acyl-CoA binding to the proteins, and changes in the tertiary structure of the enzyme were examined. The kinetics of these phenomena were related closely with each other. Furthermore, the inhibition of UGT activity was specified for acyl-CoA, though a structurally related compound, acyl-3-dephosphoCoA, had no inhibitory effect. The results suggested that the specific binding of acyl-CoA to UGT isoforms induced conformational changes of the enzymes and resultant inhibition of UGT activity.
我们之前已鉴定并纯化了尿苷二磷酸葡萄糖醛酸基转移酶（UGT）同工型，将其作为大鼠肝微粒体中蛋白质酰化作用的靶点（Yamashita等人，《生物化学杂志》312: 301 - 308, 1995）。UGT同工型的酰化作用在与酰基辅酶A孵育时发生，无需另一种蛋白质酰基转移酶，这表明这是一种自身酰化作用。该研究揭示了UGT同工型与酰基辅酶A之间的相互作用。在本研究中，利用大鼠肝微粒体和纯化的酶组分，全面研究了脂肪酰基辅酶A对UGT活性的影响。两种组分的UGT活性均受到酰基辅酶A的浓度依赖性抑制。观察到酰基辅酶A对各种底物的活性均有影响，这表明该影响显示了UGT同工型的广泛范围。为了评估酰基辅酶A抑制UGT活性的潜在机制，研究了抑制作用、酰基辅酶A与蛋白质的结合以及酶三级结构变化之间的关系。这些现象的动力学彼此密切相关。此外，虽然结构相关化合物酰基 - 3 - 去磷酸辅酶A没有抑制作用，但UGT活性的抑制作用是酰基辅酶A所特有的。结果表明，酰基辅酶A与UGT同工型的特异性结合诱导了酶的构象变化，从而导致UGT活性受到抑制。
