Rotenberg M, Zakim D
Department of Medicine, Cornell University Medical College, New York, New York 10021.
Biochemistry. 1989 Oct 17;28(21):8577-82. doi: 10.1021/bi00447a046.
The GT2P isoform of microsomal UDP-glucuronosyltransferase from pig liver is a lipid-dependent enzyme. The data in the present work indicate that, in addition to regulation of activity, the thermal stability of the enzyme also is modulated by the acyl chain composition of phosphatidylcholines (PC) used to reconstitute the activity of pure enzyme. There was a reversible, temperature-dependent change in the state of the pure enzyme to an inactive form with onset at T greater than 38 degrees C, depending on the environment of the enzyme. The midpoint for the transition shifted from 39.8 degrees C for enzyme in a bilayer of distearoylphosphatidylcholine (DSPC) to 47.5 degrees C for enzyme in a bilayer of 1-stearoyl-2-oleoylphosphatidylcholine (SOPC). For all lipids, the transition from a catalytically active to an inactive form of the enzyme was associated with large compensating changes in H and S. Lipid-induced stabilization of the active form of UDP-glucuronosyltransferase at T greater than 37 degrees C was associated with decreases in delta H and delta S, but the decreases in delta S were larger, indicating that lipid-induced stabilization of the active form of the enzyme was entropic. The transition between the active and inactive forms of the enzyme was too rapid in either direction to measure in a standard spectrophotometer. In addition to reversible inactivation of the enzyme, there was a slower irreversible, temperature-dependent inactivation. The rate of this process depended on the acyl chains of the phosphocholines interacting with the enzyme. However, there was no obvious correlation between the structures of lipids that stabilized the different inactivation reactions.
猪肝微粒体UDP-葡萄糖醛酸基转移酶的GT2P同工型是一种脂质依赖性酶。本研究中的数据表明，除了活性调节外，该酶的热稳定性还受到用于重建纯酶活性的磷脂酰胆碱（PC）酰基链组成的调节。纯酶的状态会发生可逆的、温度依赖性的变化，在T大于38℃时转变为无活性形式，这取决于酶所处的环境。转变的中点温度从二硬脂酰磷脂酰胆碱（DSPC）双层中的酶的39.8℃转变为1-硬脂酰-2-油酰磷脂酰胆碱（SOPC）双层中的酶的47.5℃。对于所有脂质，酶从催化活性形式转变为无活性形式都伴随着H和S的大幅补偿性变化。在T大于37℃时，脂质诱导的UDP-葡萄糖醛酸基转移酶活性形式的稳定与ΔH和ΔS的降低有关，但ΔS的降低幅度更大，这表明脂质诱导的酶活性形式的稳定是由熵驱动的。酶的活性形式和无活性形式之间的转变在两个方向上都太快，无法在标准分光光度计中测量。除了酶的可逆失活外，还存在较慢的不可逆的、温度依赖性失活。这个过程的速率取决于与酶相互作用的磷酸胆碱的酰基链。然而，稳定不同失活反应的脂质结构之间没有明显的相关性。
