Yoshimoto Makoto, Kozono Ryohei, Tsubomura Naoki
Department of Applied Molecular Bioscience, Yamaguchi University , 2-16-1 Tokiwadai, Ube 755-8611, Japan.
Langmuir. 2015 Jan 20;31(2):762-70. doi: 10.1021/la504126b. Epub 2015 Jan 5.
Chaperone machinery in living systems can catch denatured enzymes and induce their reactivation. Chaperone mimics are beneficial for applying enzymatic reactions in vitro. In this work, the affinity between liposomes and thermally denatured enzymes was controlled to stabilize the enzyme activity. The model enzyme is formate dehydrogenase from Candida boidinii (CbFDH) which is a homodimer and negatively charged in the phosphate buffer solution (pH 7.2) used. The activity of free CbFDH readily decreased at 58 °C following the first-order kinetics with the half-life t1/2 of 27 min. The turbidity measurements showed that the denatured enzyme molecules formed aggregates. The liposomes composed of zwitterionic phosphatidylcholines (PCs) stabilized the CbFDH activity at 58 °C, as revealed with six different PCs. The PC liposomes were indicated to bind to the aggregate-prone enzyme molecules, allowing reactivation at 25 °C. The cofactor β-reduced nicotinamide adenine dinucleotide (NADH) also stabilized the enzyme activity. The affinity between liposomes and denatured CbFDH could be modulated by incorporating cationic 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP) in PC membranes. The t1/2 values significantly increased in the presence of liposomes ([lipid] = 1.5 mM) composed of PC and DOTAP at the mole fraction f(D) of 0.1. On the other hand, the DOTAP-rich liposomes (f(D) ≥ 0.7) showed strong affinity toward denatured CbFDH, accelerating its deactivation. The liposomes with low charge density function as chaperone mimics that can efficiently catch the denatured enzymes without interfering with their intramolecular interaction for reactivation.
生物系统中的伴侣机制能够捕获变性酶并诱导其重新激活。伴侣模拟物有利于在体外应用酶促反应。在这项工作中，通过控制脂质体与热变性酶之间的亲和力来稳定酶的活性。模型酶是来自博伊丁假丝酵母的甲酸脱氢酶（CbFDH），它是一种同二聚体，在所使用的磷酸盐缓冲溶液（pH 7.2）中带负电荷。游离CbFDH的活性在58℃时遵循一级动力学迅速下降，半衰期t1/2为27分钟。浊度测量表明变性酶分子形成了聚集体。由两性离子磷脂酰胆碱（PCs）组成的脂质体在58℃时稳定了CbFDH的活性，六种不同的PCs均有此效果。PC脂质体被证明能与易于聚集的酶分子结合，使其在25℃时重新激活。辅因子β-还原型烟酰胺腺嘌呤二核苷酸（NADH）也能稳定酶的活性。通过在PC膜中掺入阳离子1,2-二油酰氧基-3-三甲基氯化铵（DOTAP），可以调节脂质体与变性CbFDH之间的亲和力。在摩尔分数f（D）为0.1的由PC和DOTAP组成的脂质体（[脂质]=1.5 mM）存在下，t1/2值显著增加。另一方面，富含DOTAP的脂质体（f（D）≥0.7）对变性CbFDH表现出很强的亲和力，加速了其失活。电荷密度低的脂质体起到伴侣模拟物的作用，能够有效地捕获变性酶，而不干扰其分子内相互作用以实现重新激活。
