Zhang L, Jackson-Machelski E, Gordon J I
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 1996 Dec 20;271(51):33131-40. doi: 10.1074/jbc.271.51.33131.
Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase (Nmt1p) is an essential 455-residue, monomeric enzyme that catalyzes the transfer of myristate from myristoyl-CoA to the NH2-terminal Gly residue of cellular proteins. Nmt1p has an ordered Bi Bi reaction mechanism with binding of myristoyl-CoA occurring before binding of peptide substrates. To define residues important for function, the polymerase chain reaction was used to generate random mutations in the NMT1 gene. A colony color sectoring assay was used to screen a library of 52,000 transformants for nmt1 alleles encoding enzymes with reduced activity. nmt1 alleles were identified that produced temperature-sensitive (ts) growth arrest due to substitutions affecting eight residues conserved in orthologous Nmts: Asn102, Ala202, Cys217, Ser328, Val395, Asn404, Leu420, and Asn426. Ala202 --> Thr, Cys217 --> Arg, Ser328 --> Pro, Asn404 --> Tyr, and Asn426 --> Ile produced the most severe ts phenotype. Their effects on the functional properties of the enzyme's myristoyl-CoA and peptide binding sites were defined by purifying each mutant from Escherichia coli and conducting in vitro kinetic analyses with acyl-CoA and peptide substrates and with two competitive inhibitors: S-(2-oxo)pentadecyl-CoA, a nonhydrolyzable myristoyl-CoA analog, and SC-58272, a peptidomimetic derived from the NH2-terminal sequence of an Nmt1p substrate (ADP-ribosylation factor-2, Arf2p). None of the substitutions affect the enzyme's acyl chain length selectivity. When compared with wild type Nmt1p, Cys217 --> Arg produces 3- and 6-fold increases in Ki for SC-58272 at 24 and 37 degrees C but no change in Ki for S-(2-oxo)pentadecyl-CoA, indicating that the substitution selectively affects Nmt1p's peptide binding site. Asn426 --> Ile selectively perturbs the myristoyl-CoA binding site, resulting in the most pronounced reduction in affinity for S-(2-oxo)pentadecyl-CoA (12- and 20-fold). Ala202 --> Thr, which confers the most severe ts phenotype, provides an example of a substitution that affects both sites, producing 3- and 6-fold increases in the Ki for S-(2-oxo)pentadecyl-CoA and 6- and 9-fold increases in the Ki for SC-58272 at 24 and 37 degrees C. An N-myristoylation-dependent change in the electrophoretic mobility of Arf1p was used to assay the effects of the mutants on cellular levels of protein N-myristoylation under a variety of growth conditions. The ts growth arrest produced by nmt1 alleles correlates with a reduction in myristoyl-Arf1p to </=50% of total cellular Arf1p.
酿酒酵母肉豆蔻酰辅酶A：蛋白质N - 肉豆蔻酰转移酶（Nmt1p）是一种由455个残基组成的必需单体酶，它催化肉豆蔻酸从肉豆蔻酰辅酶A转移到细胞蛋白质的NH2末端甘氨酸残基上。Nmt1p具有有序的双底物双分子反应机制，肉豆蔻酰辅酶A的结合先于肽底物的结合。为了确定对功能重要的残基，利用聚合酶链反应在NMT1基因中产生随机突变。使用菌落颜色扇形分析法筛选了一个包含52,000个转化体的文库，以寻找编码活性降低的酶的nmt1等位基因。鉴定出的nmt1等位基因由于影响直系同源Nmt中保守的八个残基的替代而导致温度敏感（ts）生长停滞：Asn102、Ala202、Cys217、Ser328、Val395、Asn404、Leu420和Asn426。Ala202→Thr、Cys217→Arg、Ser328→Pro、Asn404→Tyr和Asn426→Ile产生了最严重的ts表型。通过从大肠杆菌中纯化每个突变体，并使用酰基辅酶A和肽底物以及两种竞争性抑制剂进行体外动力学分析，确定了它们对酶的肉豆蔻酰辅酶A和肽结合位点功能特性的影响：S -（2 - 氧代）十五烷基辅酶A，一种不可水解的肉豆蔻酰辅酶A类似物，以及SC - 58272，一种源自Nmt1p底物（ADP - 核糖基化因子 - 2，Arf2p）NH2末端序列的拟肽。没有一个替代影响酶的酰基链长度选择性。与野生型Nmt1p相比，Cys217→Arg在24和37℃下使SC - 58272的Ki增加3倍和6倍，但S -（2 - 氧代）十五烷基辅酶A的Ki没有变化，表明该替代选择性地影响Nmt1p的肽结合位点。Asn426→Ile选择性地扰乱肉豆蔻酰辅酶A结合位点，导致对S -（2 - 氧代）十五烷基辅酶A的亲和力最明显降低（12倍和20倍）。赋予最严重ts表型的Ala202→Thr提供了一个影响两个位点的替代例子，在24和37℃下使S -（2 - 氧代）十五烷基辅酶A的Ki增加3倍和6倍，使SC - 58272的Ki增加6倍和9倍。利用Arf1p电泳迁移率的N - 肉豆蔻酰化依赖性变化来测定突变体在各种生长条件下对蛋白质N - 肉豆蔻酰化细胞水平的影响。nmt1等位基因产生的ts生长停滞与肉豆蔻酰 - Arf1p减少至总细胞Arf1p的≤50％相关。
