锤头状核酶的低温酶学
Cryoenzymology of the hammerhead ribozyme.
作者信息
Feig A L, Ammons G E, Uhlenbeck O C
机构信息
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309, USA.
出版信息
RNA. 1998 Oct;4(10):1251-8. doi: 10.1017/s1355838298980943.
Abstract
The technique of cryoenzymology has been applied to the hammerhead ribozyme in an attempt to uncover a structural rearrangement step prior to cleavage. Several cryosolvents were tested and 40% (v/v) methanol in water was found to perturb the system only minimally. This solvent allowed the measurement of ribozyme activity between 30 and -33 degrees C. Eyring plots are linear down to -27 degrees C, but a drastic reduction in activity occurs below this temperature. However, even at extremely low temperatures, the rate is still quite pH dependent, suggesting that the chemical step rather than a structural rearrangement is still rate-limiting. The nonlinearity of the Eyring plot may be the result of a transition to a cold-denatured state or a glassed state.
摘要
低温酶学技术已应用于锤头状核酶，试图揭示切割前的结构重排步骤。测试了几种低温溶剂，发现40%（v/v）的甲醇水溶液对该系统的干扰最小。这种溶剂可在30至 -33摄氏度之间测量核酶活性。艾林曲线在 -27摄氏度以下仍呈线性，但在此温度以下活性会急剧降低。然而，即使在极低温度下，反应速率仍强烈依赖于pH值，这表明化学步骤而非结构重排仍是限速步骤。艾林曲线的非线性可能是向冷变性状态或玻璃态转变的结果。
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