Identification of essential arginines in the acetate kinase from Methanosarcina thermophila.
Identification of essential arginines in the acetate kinase from Methanosarcina thermophila.
复制标题
嗜热甲烷八叠球菌乙酸激酶中必需精氨酸的鉴定。
DOI：
10.1021/bi991998h
复制
发表时间：
2000
期刊：
影响因子：
2.9
通讯作者：
Ferry,JG
中科院分区：
文献类型：
--
作者：
Singh-Wissmann,K;Miles,RD;Ingram-Smith,C;Ferry,JG
定点诱变是鉴定催化活性位点残基的有力工具；然而，这种方法直到最近才用于醋酸激酶。来自嗜热甲烷菌的酶已被克隆并在大肠杆菌（重组野生型）中以高活性形式超量生产。研究了精氨酸在该醋酸激酶中的作用。5种精氨酸（R91、R175、R241、R285、R340）。根据其在醋酸激酶同源物序列中的高度保守性，选择了嗜热酶进行单独替换。用丙氨酸或亮氨酸替代R91或R241产生的变异比活性低于重组野生型酶的0.1%。这些变异的圆二色光谱和其他特性与重组野生型相当，表明没有全局构象变化。这些结果表明R91和R241对活性是必需的，与催化作用一致。用赖氨酸保守替代R91产生的变体约有2%的重组野生型活性，这表明在这个位置带正电荷是重要的。与重组野生型相比，R91K变体的乙酸盐的km值增加了10倍以上，这表明R91在结合该底物方面发挥了额外的作用。当在反应混合物中加入胍或衍生物时，R91A和R241A变体的活性都被恢复了20倍。修复变体的ATP的km值与重组野生型相似，表明修复活性是重要官能团被替换的结果，而不是催化机制改变的结果。这些结果进一步支持了R91和R241在催化中的作用。用丙氨酸、亮氨酸或赖氨酸替代R285对活性没有显著影响；然而，乙酸酯的km值增加了6 ~ 10倍，表明R285影响了该底物的结合。重组野生型酶及其变体的苯乙二醛抑制和底物保护实验与活性位点存在一个或多个必需精氨酸残基以及R91和R241在催化中的作用一致。在ATP的γ-磷酸直接在线转移到乙酸的过程中，R91和R241起到稳定先前提出的五坐标过渡态的作用。用丙氨酸、亮氨酸或赖氨酸替代R175和R340产生的变异的动力学表征表明，这些残基不参与催化作用，但在结构上起着重要作用。
Site-directed mutagenesis is a powerful tool for identifying active-site residues essential for catalysis; however, this approach has only recently become available for acetate kinase. The enzyme fromMethanosarcina thermophilahas been cloned and hyper-produced in a highly active form inEscherichia coli(recombinant wild-type). The role of arginines in this acetate kinase was investigated. Five arginines (R91, R175, R241, R285, and R340) in theM. thermophilaenzyme were selected for individual replacement based on their high conservation among sequences of acetate kinase homologues. Replacement of R91 or R241 with alanine or leucine produced variants with specific activities less than 0.1% of the recombinant wild-type enzyme. The circular dichroism spectra and other properties of these variants were comparable to those of recombinant wild-type, indicating no global conformational changes. These results indicate that R91 and R241 are essential for activity, consistent with roles in catalysis. The variant produced by conservative replacement of R91 with lysine had approximately 2% of recombinant wild-type activity, suggesting a positive charge is important in this position. TheKmvalue for acetate of the R91K variant increased greater than 10-fold relative to recombinant wild-type, suggesting an additional role for R91 in binding this substrate. Activities of both the R91A and R241A variants were rescued 20-fold when guanidine or derivatives were added to the reaction mixture. TheKmvalues for ATP of the rescued variants were similar to those of recombinant wild-type, suggesting that the rescued activities are the consequence of replacement of important functional groups and not changes in the catalytic mechanism. These results further support roles for R91 and R241 in catalysis. Replacement of R285 with alanine, leucine, or lysine had no significant effect on activity; however, theKmvalues for acetate increased 6−10-fold, suggesting R285 influences the binding of this substrate. Phenylglyoxal inhibition and substrate protection experiments with the recombinant wild-type enzyme and variants were consistent with the presence of one or more essential arginine residues in the active site as well as with roles for R91 and R241 in catalysis. It is proposed that R91 and R241 function to stabilize the previously proposed pentacoordinate transition state during direct in-line transfer of the γ-phosphate of ATP to acetate. The kinetic characterization of variants produced by replacement of R175 and R340 with alanine, leucine, or lysine indicated that these residues are not involved in catalysis but fulfill important structural roles.