Ogasahara K, Nakamura M, Nakura S, Tsunasawa S, Kato I, Yoshimoto T, Yutani K
Institute for Protein Research, Osaka University, Japan.
Biochemistry. 1998 Dec 15;37(50):17537-44. doi: 10.1021/bi9814585.
To elucidate the energetic features of the anomalously high-level stabilization of a hyperthermophile pyrrolidone carboxyl peptidase (PfPCP) from a hyperthermophilic archaeon, Pyrococcus furiosus, equilibrium and kinetic studies of the guanidine hydrochloride (GuHCl)-induced unfolding and refolding were carried out with CD measurements at 220 nm in comparison with those from the mesophile homologue (BaPCP) from Bacillus amyloliquefaciens. The mutant protein of PfPCP substituted with Ser at both Cys142 and Cys188 (PfC142/188S) was used. The GuHCl unfolding for PfC142/188S and BaPCP was reversible. It was difficult to obtain the equilibrated unfolding curve of the hyperthermophile proteins at temperatures below 50 degreesC and pH 7, because of the remarkably slow rate of the unfolding. The unfolding for PfC142/188S attained equilibrium after 7 days at 60 degreesC, resulting in the coincidence between the unfolding and refolding curves. The Gibbs energy change of unfolding, DeltaGH2O (56.6 kJ/mol), for PfC142/188S at 60 degreesC and pH 7 was dramatically higher than that (7.6 kJ/mol) for BaPCP at 40 degreesC and pH 7. The unfolding and refolding kinetics for PfC142/188S and BaPCP at both 25 and 60 degreesC at pH 7 were approximated as a single exponential. The rate constant in water (kuH2O) of the unfolding reaction for PfC142/188S (1.6 x 10(-)15 s-1) at 25 degreesC and pH 7 was drastically reduced by 7 orders of magnitude compared to that (1.5 x 10(-)8 s-1) for BaPCP, whereas the refolding rates (krH2O) in water for PfC142/188S (9.3 x 10(-)2 s-1) and BaPCP (3.6 x 10(-)1 s-1) at 25 degreesC and pH 7 were similar. These results indicate that the greater stability of the hyperthermophile PCP was characterized by the drastically slow unfolding rate.
为阐明嗜热古菌激烈火球菌（Pyrococcus furiosus）中嗜热嗜热菌吡咯烷酮羧基肽酶（PfPCP）异常高水平稳定化的能量特征，通过在220 nm处进行圆二色性（CD）测量，对盐酸胍（GuHCl）诱导的解折叠和重折叠进行了平衡和动力学研究，并与解淀粉芽孢杆菌（Bacillus amyloliquefaciens）嗜温同源物（BaPCP）的研究结果进行了比较。使用了在Cys142和Cys188处均被丝氨酸取代的PfPCP突变蛋白（PfC142/188S）。PfC142/188S和BaPCP的GuHCl解折叠是可逆的。由于解折叠速率非常缓慢，在温度低于50℃和pH 7的条件下，很难获得嗜热菌蛋白的平衡解折叠曲线。PfC142/188S在60℃下7天后达到解折叠平衡，导致解折叠曲线和重折叠曲线重合。在60℃和pH 7条件下，PfC142/188S的解折叠吉布斯自由能变化ΔGH2O（56.6 kJ/mol）显著高于在40℃和pH 7条件下BaPCP的ΔGH2O（7.6 kJ/mol）。在25℃和60℃、pH 7条件下，PfC142/188S和BaPCP的解折叠和重折叠动力学近似为单指数形式。在25℃和pH 7条件下，PfC142/188S解折叠反应在水中的速率常数（kuH2O）（1.6×10−15 s−1）与BaPCP的速率常数（1.5×10−8 s−1）相比急剧降低了7个数量级，而在25℃和pH 7条件下，PfC142/188S（9.3×10−2 s−1）和BaPCP（3.6×10−1 s−1）在水中的重折叠速率（krH2O）相似。这些结果表明，嗜热菌PCP更高的稳定性表现为解折叠速率急剧减慢。
