IDENTIFICATION OF AN ACTIVE-SITE ARGININE IN RAT CHOLINE-ACETYLTRANSFERASE BY ALANINE SCANNING MUTAGENESIS
IDENTIFICATION OF AN ACTIVE-SITE ARGININE IN RAT CHOLINE-ACETYLTRANSFERASE BY ALANINE SCANNING MUTAGENESIS
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DOI：
10.1074/jbc.270.49.29111
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发表时间：
1995-12-08
影响因子：
4.8
通讯作者：
HERSH, LB
中科院分区：
文献类型：
--
作者：
WU, DH;HERSH, LB
动力学和化学修饰研究表明胆碱乙酰转移酶中存在活性位点精氨酸，其功能是通过与辅酶 A 底物的 5'-磷酸相互作用来稳定辅酶结合。为了鉴定该残基，通过定点诱变将大鼠胆碱乙酰转移酶中的七个保守精氨酸转化为丙氨酸，并将这些突变体的特性与野生型酶进行比较。用丙氨酸取代精氨酸 452 导致 CoA 和乙酰胆碱以及 K-cat 的 K-m 增加 7-12 倍，而去磷酸-CoA 的 K-m 几乎没有变化。产物抑制研究表明胆碱是乙酰胆碱的竞争性抑制剂，表明 R452A 遵循与野生型酶相同的 Theorell-Chance 动力学机制。 R452Q 和 R452E 获得了类似的结果，后者显示出动力学参数的最大变化。这些发现与 Arg-452 突变增加了辅酶从酶解离的速率常数 k(5) 一致。通过显示 R452A 突变体对辅酶 A 的亲和力降低 5-10 倍，获得了精氨酸 452 参与辅酶 A 结合的直接证据，这通过防止苯基乙二醛失活以及热失活的能力来确定。
Kinetic as well as chemical modification studies have implicated the presence of an active site arginine in choline acetyltransferase, whose function is to stabilize coenzyme binding by interacting with the 5'-phosphate of the coenzyme A substrate. In order to identify this residue seven conserved arginines in rat choline acetyltransferase were converted to alanine by site-directed mutagenesis, and the properties of these mutants were compared with the wild type enzyme. Substitution of arginine 452 with alanine resulted in a 7-12-fold increase in the K-m for both CoA and acetylcholine as well as K-cat with little change in the K-m for dephospho-CoA. Product inhibition studies showed choline to be a competitive inhibitor with respect to acetylcholine, indicating R452A follows the same Theorell-Chance kinetic mechanism as the wild type enzyme. Similar results were obtained with R452Q and R452E, with the latter showing the largest changes in kinetic parameters, These findings are consistent with Arg-452 mutations increasing the rate constant, k(5), for dissociation of the coenzyme from the enzyme. Direct evidence that arginine 452 is involved in coenzyme A binding was obtained by showing a 5-10-fold decrease in affinity of the R452A mutant for coenzyme A as determined by the ability to protect against phenylglyoxal inactivation as well as thermal inactivation.