EDITING MECHANISM FOR THE METHIONYL-TRANSFER RNA-SYNTHETASE IN THE SELECTION OF AMINO-ACIDS IN PROTEIN-SYNTHESIS
EDITING MECHANISM FOR THE METHIONYL-TRANSFER RNA-SYNTHETASE IN THE SELECTION OF AMINO-ACIDS IN PROTEIN-SYNTHESIS
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DOI：
10.1021/bi00574a021
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发表时间：
1979-01-01
期刊：
影响因子：
2.9
通讯作者：
DINGWALL, C
中科院分区：
文献类型：
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作者：
FERSHT, AR;DINGWALL, C
商业氨基酸样品中的微量蛋氨酸杂质掩盖了甲硫酰-tRNA合成酶催化的失活模式，并掩盖了编辑机制的存在。α-氨基丁酸酯、缬氨酸和异亮氨酸的重结晶样品被发现在嗜热脂肪芽孢杆菌的酶催化下促进焦磷向三磷酸腺苷的交换，Kcat(催化速率常数)值接近蛋氨酸刺激的反应。在用Raney镍处理从非特异性氨基酸中去除蛋氨酸之后，活性被取消，支化的侧链氨基酸被某些未支化的侧链氨基酸显著减少。有一种编辑机制可以去除侧链比蛋氨酸更小的氨基酸的失活：来自大肠杆菌和嗜热脂肪杆菌的酶不催化同型半胱氨酸或去甲伐氨酸向tRNAMet的转移，而是催化ATP焦磷酸酶的活性。在同型半胱氨酸和tRNAMet存在下，ATP焦磷酸酶活性的转换数与tRNA与蛋氨酸的氨酰化反应的转换数相似。氨基酸按大小排序，符合双筛编辑机制。比蛋氨酸空间较小的氨基酸被激活，kcat/Km的值降低，其产物通过水解性编辑被去除。空间上较大的乙硫氨酸被激活，其产物耐水解。去甲亮氨酸虽然名义上与蛋氨酸是异构性的，但略小，即将被编辑：既有向tRNAMet的转移，也有氨基酸刺激的ATP/焦磷酸酶活性。TRNAMet的不接受末端羟基不是编辑反应所必需的。合成的tRNAMet在这个位置上是脱氧的，不接受同型半胱氨酸或去甲戊氨酸，但刺激氨基酸依赖的ATP焦磷酸酶反应。
Trace impurities of methionine in commercial samples of amino acids obscured the pattern of misactivations catalyzed by methionyl-tRNA synthetases and concealed the existence of an editing mechanism. Recrystallized samples of .alpha.-aminobutyrate, valine and isoleucine are found to stimulate the exchange of pyrophosphate into ATP catalyzed by the enzyme from Bacillus stearothermophilus with values of Kcat (catalytic rate constant) approaching that for the methionine-stimulated reaction. Following removal of methionine from the nonspecific amino acids by treatment with Raney nickel, activity is abolished with the branched side chain amino acids considerably reduced with certain unbranched side chain amino acids. There is an editing mechanism for the removal of the misactivation of amino acids with smaller side chains than methionine: the enzymes from Escherichia coli and B. stearothermophilus do not catalyze the transfer of homocysteine or norvaline to tRNAMet but, instead, catalyze an ATP-pyrophosphatase activity. The turnover number for the ATP-pyrophosphatase activity in the presence of homocystein and tRNAMet is similar to that for the aminoacylation of the tRNA with methionine. The amino acids are sorted by size in accord with the double-sieve editing mechanism. Amino acids sterically smaller than methionine are activated with reduced values of kcat/Km and their products are removed by hydrolytic editing. The sterically larger ethionine is activated and its products are resistant to hydrolysis. Norleucine, which although nominally isosteric with methionine is slightly smaller, is on the borderline of being edited: there is both transfer to tRNAMet and an amino acid stimulated ATP/pyrophosphatase activity. The nonaccepting terminal hydroxyl group of tRNAMet is not essential for the editing reaction. The synthetic tRNAMet which is deoxy in this position does not accept homocysteine or norvaline but stimulates the amino acid dependent ATP-pyrophosphatase reaction.