Guez-Ivanier V, Hermann M, Baldwin D, Bedouelle H
Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
J Mol Biol. 1993 Nov 5;234(1):209-21. doi: 10.1006/jmbi.1993.1575.
The tyrosyl-tRNA synthetases from Bacillus stearothermophilus (Bst-TyrTS) and Escherichia coli (Eco-TyrTS) are 56% identical in amino acid sequence. To map and characterize the set of interactions that makes Bst-TyrTS more stable than Eco-TyrTS, a family of nine hybrid proteins was constructed between the two enzymes. The N-terminal part of each hybrid came from Eco-TyrTS and the C-terminal part from Bst-TyrTS. The stability and activity of these hybrids were estimated by experiments of thermal inactivation and tRNA charging. For all the hybrids, the temperature of half-inactivation in 30 minutes was above 44 degrees C and the rate of charging was at least 40% that of Bst-TyrTS. In general, the temperature of half-inactivation increased and the rate of charging decreased monotonically when the number of residues coming from the more stable and less active Bst-TyrTS increased. As a result, the rate of charging decreased when the temperature of half-inactivation increased. These results show that the sequences and structures of the two enzymes can replace each other locally and still give a stable and active TyrTS, and that the greater stability of Bst-TyrTS is due to cumulative changes of residues scattered along the sequence. They suggest that Bst-TyrTS is more rigid than Eco-TyrTS at low temperature. The existence of a few exceptional hybrids, having stabilities or activities lower than those of the neighbouring hybrids, shows that compensatory changes of residues have occurred between the two sequences during evolution. These exceptions could be explained by the systematic identification of the couples of residues that are in contact in the Bst-TyrTS structure and become heterologous in some hybrids.
嗜热脂肪芽孢杆菌（Bst-TyrTS）和大肠杆菌（Eco-TyrTS）的酪氨酰-tRNA合成酶的氨基酸序列有56%的同源性。为了定位和表征使Bst-TyrTS比Eco-TyrTS更稳定的相互作用组，构建了一个由这两种酶之间的九种杂合蛋白组成的家族。每个杂合蛋白的N端部分来自Eco-TyrTS，C端部分来自Bst-TyrTS。通过热失活实验和tRNA充电实验估计了这些杂合蛋白的稳定性和活性。对于所有杂合蛋白，30分钟内半失活温度高于44℃，充电速率至少是Bst-TyrTS的40%。一般来说，当来自更稳定但活性较低的Bst-TyrTS的残基数量增加时，半失活温度升高，充电速率单调下降。结果，半失活温度升高时充电速率下降。这些结果表明，两种酶的序列和结构可以在局部相互替代，仍然产生稳定且有活性的TyrTS，并且Bst-TyrTS的更高稳定性归因于沿序列分散的残基的累积变化。它们表明Bst-TyrTS在低温下比Eco-TyrTS更刚性。存在一些稳定性或活性低于相邻杂合蛋白的异常杂合蛋白，这表明在进化过程中两个序列之间发生了残基的补偿性变化。这些例外情况可以通过系统鉴定在Bst-TyrTS结构中相互接触且在某些杂合蛋白中变为异源的残基对来解释。
