Wu D, Hersh L B
Department of Biochemistry, University of Kentucky, Lexington 40536-0084, USA.
J Biol Chem. 1995 Dec 8;270(49):29111-6. doi: 10.1074/jbc.270.49.29111.
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 3'-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 Km for both CoA and acetylcholine as well as kcat, with little change in the Km 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, k5, 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.
动力学以及化学修饰研究表明，胆碱乙酰转移酶中存在一个活性位点精氨酸，其功能是通过与辅酶A底物的3'-磷酸相互作用来稳定辅酶结合。为了鉴定这个残基，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，并将这些突变体的性质与野生型酶进行比较。用丙氨酸取代精氨酸452导致辅酶A和乙酰胆碱的Km以及kcat增加7至12倍，而脱磷酸辅酶A的Km变化很小。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也得到了类似的结果，后者在动力学参数上变化最大。这些发现与精氨酸452突变增加辅酶从酶上解离的速率常数k5一致。通过显示R452A突变体对辅酶A的亲和力降低5至10倍，获得了精氨酸452参与辅酶A结合的直接证据，这是通过防止苯乙二醛失活以及热失活的能力来确定的。
