Rudnick D A, McWherter C A, Adams S P, Ropson I J, Duronio R J, Gordon J I
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1990 Aug 5;265(22):13370-8.
Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase has been efficiently expressed in Escherichia coli and subsequently purified to homogeneity using phosphocellulose chromatography. The interactions between apoenzyme and its acyl-CoA and peptide ligands were examined by an isoelectric focusing gel shift assay, circular dichroism, and fluorescence spectroscopy, and a continuous assay of enzyme activity which measures the release of CoA from acyl-CoA using the thiol-specific reagent 5-5'-dithiobis-2-nitrobenzoate. Addition of myristoyl-CoA (without a substrate peptide) results in the formation of a high affinity reaction intermediate which can be operationally defined by the appearance of a more acidic enzyme isoform and by quenching of the tryptophan emission with a maximal difference at 340 nm. Circular dichroism spectroscopy indicates that these changes are accompanied by minimal changes in the enzyme's secondary structure. Incubation of purified NMT with [1-14C] myristoyl-CoA, followed by chymotryptic digestion, denaturing polyacrylamide gel electrophoresis, and treatment with hydroxylamine yielded results that are highly suggestive of a covalent ester-linked acyl-enzyme complex. Edman degradation of chymotryptic peptides has narrowed the site of interaction to a domain spanning Arg42 to Thr220 of the 455 amino acid acyltransferase. An octapeptide containing Gly but not Ala at position 1 is able to reverse the change in pI and reduce the quenching almost entirely. These data suggest a preferred order or ping-pong reaction mechanism with the acyl-CoA substrate binding event occurring first. They also indicate that Gly1 is absolutely necessary for the reaction to proceed forward from the acyl-enzyme reaction intermediate.
酿酒酵母肉豆蔻酰辅酶A：蛋白质N - 肉豆蔻酰转移酶已在大肠杆菌中高效表达，随后通过磷酸纤维素色谱法纯化至同质。通过等电聚焦凝胶迁移分析、圆二色性和荧光光谱法，以及使用硫醇特异性试剂5,5'-二硫代双-2-硝基苯甲酸测量酰基辅酶A中辅酶A释放的酶活性连续测定法，研究了脱辅酶与其酰基辅酶A和肽配体之间的相互作用。添加肉豆蔻酰辅酶A（无底物肽）会导致形成高亲和力反应中间体，其可通过更酸性的酶同工型的出现以及色氨酸发射在340 nm处的最大差异淬灭来操作定义。圆二色光谱表明这些变化伴随着酶二级结构的最小变化。用[1-14C]肉豆蔻酰辅酶A孵育纯化的NMT，然后进行胰凝乳蛋白酶消化、变性聚丙烯酰胺凝胶电泳和羟胺处理，得到的结果强烈暗示共价酯连接的酰基酶复合物。胰凝乳蛋白酶肽段的埃德曼降解已将相互作用位点缩小到455个氨基酸酰基转移酶中跨越Arg42至Thr220的结构域。在位置1含有Gly但不含Ala的八肽能够逆转pI的变化并几乎完全减少淬灭。这些数据表明了一种优先顺序或乒乓反应机制，其中酰基辅酶A底物结合事件首先发生。它们还表明Gly1对于反应从酰基酶反应中间体向前进行绝对必要。
