Shin I, Silman I, Bon C, Weiner L
Department of Neurobiology and Chemical Services, Weizmann Institute of Science, Rehovot 76100, Israel.
Biochemistry. 1998 Mar 31;37(13):4310-6. doi: 10.1021/bi973005q.
The kinetics of thermal inactivation of acetylcholinesterase from the venom of the snake, Bungarus fasciatus, were studied at 45-54 degrees C. An Arrhenius plot reveals an activation energy of 113 kcal/mol. The thermally denatured enzyme displays the spectroscopic characteristics of a partially unfolded 'molten globule' state. The rate of thermal denaturation is greatly enhanced in the presence of unilamellar vesicles of dimyristoylphosphatidylcholine, the energy barrier for the transition being lowered from 113 to 52 kcal/mol. In contrast to our findings for partially unfolded Torpedo californica acetylcholinesterase [Shin et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 2848-2852], the thermally denatured snake enzyme does not remain bound to the liposomes but is released after unfolding and subsequently aggregates. The liposomes thus serve as catalysts for unfolding of the snake enzyme, and its rate of unfolding in the presence of liposomes can be described by the Michaelis-Menten equation (Km = 8 x 10(-7) M). The phospholipid vesicles display a catalytic turnover number of kcat approximately 4 min-1, assuming 15 binding sites per vesicle for the snake acetylcholinesterase.
在45 - 54摄氏度下研究了银环蛇毒液中乙酰胆碱酯酶的热失活动力学。阿累尼乌斯曲线显示活化能为113千卡/摩尔。热变性酶呈现出部分展开的“熔球”状态的光谱特征。在二肉豆蔻酰磷脂酰胆碱单层囊泡存在下，热变性速率大大提高，转变的能量屏障从113千卡/摩尔降至52千卡/摩尔。与我们对部分展开的加州电鳐乙酰胆碱酯酶的研究结果[Shin等人（1997年），美国国家科学院院刊94，2848 - 2852]相反，热变性的蛇酶不会保持与脂质体结合，而是在展开后释放并随后聚集。因此，脂质体作为蛇酶展开的催化剂，其在脂质体存在下的展开速率可以用米氏方程描述（Km = 8×10⁻⁷ M）。假设每个脂质体有15个蛇乙酰胆碱酯酶结合位点，磷脂囊泡的催化周转数kcat约为4分钟⁻¹。
