Singh-Wissmann K, Miles R D, Ingram-Smith C, Ferry J G
Department of Biochemistry and Molecular Biology, Eberly College of Science, The Pennsylvania State University, University Park, Pennsylvania 16802-4500, USA.
Biochemistry. 2000 Apr 4;39(13):3671-7. doi: 10.1021/bi991998h.
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 from Methanosarcina thermophila has been cloned and hyper-produced in a highly active form in Escherichia coli (recombinant wild-type). The role of arginines in this acetate kinase was investigated. Five arginines (R91, R175, R241, R285, and R340) in the M. thermophila enzyme 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. The K(m) value 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. The K(m) values 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, the K(m) values 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 gamma-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.
定点诱变是一种用于鉴定催化过程中必需的活性位点残基的强大工具；然而，这种方法直到最近才应用于乙酸激酶。嗜热甲烷八叠球菌的这种酶已被克隆，并在大肠杆菌中以高活性形式大量表达（重组野生型）。研究了精氨酸在这种乙酸激酶中的作用。基于嗜热甲烷八叠球菌酶中5个精氨酸（R91、R175、R241、R285和R340）在乙酸激酶同源物序列中的高度保守性，选择它们进行逐个替换。用丙氨酸或亮氨酸替换R91或R241产生的变体的比活性低于重组野生型酶的0.1%。这些变体的圆二色光谱和其他性质与重组野生型相当，表明没有全局构象变化。这些结果表明R91和R241对活性至关重要，这与它们在催化中的作用一致。用赖氨酸保守替换R91产生的变体具有约2%的重组野生型活性，表明该位置带正电荷很重要。R91K变体对乙酸的K(m)值相对于重组野生型增加了10倍以上，表明R91在结合该底物中还有额外作用。当向反应混合物中加入胍或其衍生物时，R91A和R241A变体的活性提高了20倍。拯救后变体对ATP的K(m)值与重组野生型相似，表明拯救后的活性是重要官能团被替换的结果，而非催化机制发生了变化。这些结果进一步支持了R91和R241在催化中的作用。用丙氨酸、亮氨酸或赖氨酸替换R285对活性没有显著影响；然而，对乙酸的K(m)值增加了6至10倍，表明R285影响该底物的结合。对重组野生型酶和变体进行苯乙二醛抑制和底物保护实验，结果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。有人提出，R91和R241的作用是在ATP的γ - 磷酸基团直接向乙酸进行线性转移过程中稳定先前提出的五配位过渡态。用丙氨酸、亮氨酸或赖氨酸替换R175和R340产生的变体的动力学特征表明，这些残基不参与催化，但起着重要的结构作用。
