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首页> 外文期刊>Biochemistry >KINETICS AND THERMODYNAMICS OF INTERMOLECULAR CATALYSIS BY HAIRPIN RIBOZYMES
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KINETICS AND THERMODYNAMICS OF INTERMOLECULAR CATALYSIS BY HAIRPIN RIBOZYMES

机译：用发夹状核酶进行分子间催化的动力学和热力学

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The hairpin ribozyme, derived from the negative strand of the satellite RNA of tobacco ringspot virus, belongs to the class of small catalytic RNAs that cleave RNA to generate 2',3'-cyclic phosphate and 5'-hydroxyl termini and ligate these termini in the reverse reaction to form 3',5'-phosphodiesters. Rate and equilibrium constants for binding, dissociation, cleavage, and ligation steps in the kinetic mechanism were determined using a series of hairpin ribozyme/substrate pairs that differed in the sequence and length of the intermolecular base-paired helices. All hairpin variants cleaved with rate constants of similar to 0.3 min(-1) at pH 7.5 in 10 mM MgCl2 at 25 degrees C, regardless of the length or sequence of the intermolecular helices. A rate constant of similar to 3 min(-1) was determined for an intermolecular ligation reaction in which both cleavage products were supplied to the ribozyme in trans. Thus, the hairpin favored ligation over cleavage by 10-fold when the ribozyme was saturated with cleavage products. Binding rate constants for cleavage substrates and products were comparable to values reported for other catalytic RNAs but were somewhat slower than binding rates typical of small RNA helices. Substrate dissociation rate constants were much slower than cleavage rate constants for all substrates. Because virtually every substrate that was bound was cleaved before it could dissociate, K-M(S) values were not the same as K-d(S) values. Instead, K-M(S) reflected the ratio of cleavage and substrate binding rate constants and had the same value of similar to 30 nM for all substrates. Calculations based on empirically determined free energy parameters for simple RNA helices indicated that complexes between ribozymes and 5'-cleavage products were slightly less stable than simple helices with the same sequences. In contrast, affinities between ribozymes and cleavage substrates and between ribozymes and 3'-cleavage products were stronger than expected for simple duplexes by about -2.5 kcal/mol, evidence of stabilizing interactions in addition to those contributed by helical base pairs. This kinetic and thermodynamic study demonstrates that the kinetic mechanism of the hairpin ribozyme is distinct from the kinetic mechanisms of other well-characterized ribozymes and provides a foundation for further exploration of the hairpin structure and catalytic mechanism.
机译：发夹状核酶，来源于烟草环状斑点病毒卫星RNA的负链，属于一类小催化RNA，可裂解RNA产生2'，3'-环磷酸和5'-羟基末端，并连接这些末端。逆反应形成3'，5'-磷酸二酯。使用一系列发夹状核酶/底物对来确定动力学机制中结合，解离，裂解和连接步骤的速率和平衡常数，这些发夹状核酶/底物对的分子间碱基配对螺旋的序列和长度不同。所有发夹变体均在25摄氏度下于10 mM MgCl2中于pH 7.5在pH 7.5时以类似于0.3 min（-1）的速率常数裂解，无论分子间螺旋的长度或序列如何。分子间连接反应的速率常数与3 min（-1）相似，其中两个裂解产物均反式提供给核酶。因此，当核酶被切割产物饱和时，发夹使连接比切割更容易连接10倍。裂解底物和产物的结合速率常数与其他催化RNA报道的值相当，但比小RNA螺旋的典型结合速率慢。底物解离速率常数比所有底物的裂解速率常数慢得多。因为实际上每个结合的底物在解离之前都已裂解，所以K-M（S）值与K-d（S）值不同。取而代之的是，K-M（S）反映了裂解的比率和底物结合速率常数，并且对于所有底物，其具有类似于30 nM的相同值。基于经验确定的简单RNA螺旋的自由能参数的计算表明，核酶和5'裂解产物之间的复合物比具有相同序列的简单螺旋的稳定性稍差。相反，核酶和裂解底物之间以及核酶和3'-裂解产物之间的亲和力比简单双链体的预期亲和力强约-2.5 kcal / mol，除了螺旋碱基对产生的稳定相互作用的证据。动力学和热力学研究表明，发夹状核酶的动力学机理与其他特征明​​确的核酶的动力学机理不同，并为进一步探索发夹结构和催化机理提供了基础。

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