Bevers S, Ha S B, McLaughlin L W
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, USA.
Biochemistry. 1999 Jun 15;38(24):7710-8. doi: 10.1021/bi982977a.
Three modified hammerhead ribozyme/substrate complexes have been prepared in which individual uridine O2-carbonyls have been eliminated. The modified complexes were chemically synthesized with the substitution of a single 2-pyridone (2P) base analogue for residues U4, U7, and U16.1. Steady-state kinetic analyses indicate that the cleavage efficiencies for the U7 and U16.1 complexes were not significantly reduced relative to the native complex as measured by kcat/KM. The cleavage efficiency for the 2P4 complex, with the analogue present within the uridine loop, was reduced by greater than 2 orders of magnitude. This significant reduction in catalytic efficiency was due primarily to a decrease in kcat. The pH vs cleavage rate profile suggests that the O2-carbonyl of the U4 residue of the hammerhead complex is critical for transition state stabilization and efficient cleavage activity. The results of a Mg2+ rescue assay do not implicate the O2-carbonyl of U4 in an interaction with a divalent metal ion. In addition, the results of a ribozyme folding assay suggest that the presence of the 2P4 within the uridine loop does not alter the folding pathway (relative to the native sequence) both in the absence and in the presence of Mg2+. The O2-carbonyl of U4 appears oriented toward the interior of the catalytic pocket where it may be involved in a critical hydrogen bonding interaction necessary for transition state stabilization.
已制备出三种修饰的锤头状核酶/底物复合物，其中单个尿苷的O2-羰基已被去除。这些修饰的复合物通过化学合成，用单个2-吡啶酮（2P）碱基类似物取代U4、U7和U16.1残基。稳态动力学分析表明，通过kcat/KM测量，U7和U16.1复合物的切割效率相对于天然复合物没有显著降低。尿苷环内存在类似物的2P4复合物的切割效率降低了两个以上数量级。催化效率的显著降低主要是由于kcat的降低。pH与切割速率曲线表明，锤头状复合物U4残基的O2-羰基对于过渡态稳定和高效切割活性至关重要。Mg2+拯救试验的结果并未表明U4的O2-羰基与二价金属离子相互作用。此外，核酶折叠试验的结果表明，尿苷环内2P4的存在在不存在和存在Mg2+的情况下均不会改变折叠途径（相对于天然序列）。U4的O2-羰基似乎朝向催化口袋内部，在那里它可能参与过渡态稳定所需的关键氢键相互作用。
