Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase.
Purification and characterization of yeast myristoyl CoA:protein N-myristoyltransferase.
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DOI：
10.1073/pnas.84.9.2708
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发表时间：
1987-05
影响因子：
11.1
通讯作者：
D. Towler;S. Adams;S. R. Eubanks;D. S. Towery;E. Jackson-Machelski;L. Glaser;J. Gordon
中科院分区：
文献类型：
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作者：
D. Towler;S. Adams;S. R. Eubanks;D. S. Towery;E. Jackson-Machelski;L. Glaser;J. Gordon
肉豆蔻酰辅酶A：蛋白N-肉豆蔻酰转移酶（NMT）催化肉豆蔻酸添加到许多真核蛋白的氨基末端甘氨酸残基。最近，我们开发了一种用于分析NMT活性的无细胞系统，并开始使用一系列合成肽来表征该酶的底物特异性。我们现在已经从酿酒酵母中纯化了NMT至表观均一性。天然酶是一种55 kDa的蛋白质，表现出不需要二价阳离子，似乎含有一个组氨酸残基的酶活性的关键。总共有42种合成肽已被用于确定NMT底物的结构/活性关系。酰化需要氨基末端甘氨酸;用甘氨酸类似物取代产生作为NMT底物或抑制剂无活性的肽。在位置3和4处允许氨基酸的广谱，而在位置5处显示严格的氨基酸要求。用Asp取代肽Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg中的Ala 5消除了肽的肉豆蔻酰接受活性。在三个不同序列的情况下，在该位置的丝氨酸导致表观Km降低约等于500倍。五肽和六肽是底物，但亲和力降低。这些研究确定了对NMT-配体相互作用重要的结构信息存在于肽底物中的前两个氨基酸之外，并且残基5的侧链在底物与该酶的结合中起关键作用。
Myristoyl CoA:protein N-myristoyltransferase (NMT) catalyzes the addition of myristic acid to the amino-terminal glycine residues of a number of eukaryotic proteins. Recently, we developed a cell-free system for analyzing NMT activity and have begun to characterize the substrate specificity of this enzyme by using a series of synthetic peptides. We have now purified NMT from Saccharomyces cerevisiae to apparent homogeneity. The native enzyme is a 55-kDa protein, exhibits no requirement for divalent cation, and appears to contain a histidine residue critical for enzyme activity. A total of 42 synthetic peptides have been used to define structure/activity relationships in NMT substrates. An amino-terminal glycine is required for acylation; substitution with glycine analogues produces peptides that are inactive as substrates or inhibitors of NMT. A broad spectrum of amino acids is permitted at positions 3 and 4, while strict amino acid requirements are exhibited at position 5. Replacement of Ala5 in the peptide Gly-Asn-Ala-Ala-Ala-Ala-Arg-Arg with Asp ablates the peptide's myristoyl-accepting activity. A serine at this position results in a decrease by a factor of approximately equal to 500 in the apparent Km in the context of three different sequences. Penta- and hexa-peptides are substrates, but with decreased affinity. These studies establish that structural information important for NMT-ligand interaction exists beyond the first two amino acids in peptide substrates and that the side chains of residue 5 play a critical role in the binding of substrates to this enzyme.