McLeod Aileen C, Lilley David M J
Cancer Research UK Nucleic Acid Structure Research Group, Department of Biochemistry, MSI/WTB Complex, The University of Dundee, UK.
Biochemistry. 2004 Feb 3;43(4):1118-25. doi: 10.1021/bi035790e.
The VS ribozyme acts as a very efficient ligase in trans when the 5' cleavage product is prevented from dissociation by an extended helix Ia in the substrate. Provided that the length of this helix is >or=10 bp, the substrate becomes approximately 80% ligated by the ribozyme acting in trans. Most of the nucleotides that have been shown to be important for cleavage are similarly important for ligation, including the critical A756 of the active site. The exception to this is C755. The variant ribozyme C755A has almost normal cleavage activity, whereas the rate of ligation is reduced 70-fold. It is therefore likely that this nucleotide plays a specific role in the organization of the termini of the ligation substrates. We have found that the rate of the trans ligation reaction depends on pH, corresponding to the protonation/deprotonation of a group with a pK(A) of 5.6. A model is suggested whereby the approach to equilibrium is catalyzed by the ribozyme catalyzing the ligation reaction in its deprotonated state (rate 1.05 min(-1)) and the cleavage reaction in its protonated state (rate 0.18 min(-1)). A756 is a candidate for the nucleobase undergoing protonation/deprotonation.
当5'切割产物因底物中延伸的螺旋Ia而无法解离时，VS核酶在反式作用中可作为一种非常有效的连接酶。只要该螺旋的长度≥10个碱基对，底物通过反式作用的核酶进行连接的比例约为80%。已证明对切割重要的大多数核苷酸对连接同样重要，包括活性位点关键的A756。C755是个例外。变体核酶C755A具有几乎正常的切割活性，而连接速率降低了70倍。因此，该核苷酸可能在连接底物末端的组织中发挥特定作用。我们发现反式连接反应的速率取决于pH值，这与pK(A)为5.6的基团的质子化/去质子化相对应。提出了一个模型，据此平衡的趋近由处于去质子化状态催化连接反应（速率为1.05 min(-1)）和处于质子化状态催化切割反应（速率为0.18 min(-1)）的核酶催化。A756是发生质子化/去质子化的核碱基的候选者。
