McIlhinney R A, Patel P B, McGlone K
Medical Research Council Anatomical Neuropharmacology Unit, Oxford, England.
Eur J Biochem. 1994 May 15;222(1):137-46. doi: 10.1111/j.1432-1033.1994.tb18851.x.
The enzyme myristoyl-CoA:protein N-myristoyltransferase is responsible for the attachment of a myristoyl group to the N-terminal glycine of a number of cell, viral and fungal proteins. In order to overcome the difficulties of purification of this enzyme from tissue sources, we have produced an N-terminally polyhistidine-tagged version of the enzyme and expressed this in Escherichia coli. The resulting enzyme has a molecular mass of 53 kDa and is fully active showing the expected specificity for myristic acid and causing the N-terminal myristoylation of both synthetic peptide and protein substrates in vitro. The enzyme exhibits a broad pH optimum peaking at a pH of 8.0 and has a Km for myristoyl-CoA of 7.6 microM. The two synthetic peptide substrates based on the N-terminal sequence of the catalytic subunit of protein kinase A (GNAAAARR) and of p60src (GSSKSKPKDPSQRRRY) have different kinetic parameters with Km values of 115.2 microM and 44.2 microM and Vmax values of 95 and 120 nmol.min-1.mg-1, respectively. The expressed enzyme is partially inhibited (50%) by iodoacetamide at 5 mM and fully inhibited by diethylpyrocarbonate at 10 mM. This latter inhibition can be prevented by including histidine in the incubation of the enzyme and inhibitor. Antisera raised to synthetic peptides based on sequences derived from the N- and C- terminus of the human enzyme reacted with the expressed protein on Western blots, but only the N-terminal sequence reacted with the native protein suggesting that the C-terminus may be not be accessible. The enzyme can catalyse the removal of a myristoyl group from myristoylated peptides but does so only in the presence of added coenzyme A.
肉豆蔻酰辅酶A：蛋白质N-肉豆蔻酰基转移酶负责将肉豆蔻酰基连接到许多细胞、病毒和真菌蛋白质的N端甘氨酸上。为了克服从组织来源纯化这种酶的困难，我们制备了一种N端带有多组氨酸标签的该酶版本，并在大肠杆菌中进行表达。所得酶的分子量为53 kDa，具有完全活性，对肉豆蔻酸表现出预期的特异性，并在体外导致合成肽和蛋白质底物的N端肉豆蔻酰化。该酶在pH 8.0时表现出较宽的最适pH值，肉豆蔻酰辅酶A的Km值为7.6 μM。基于蛋白激酶A催化亚基（GNAAAARR）和p60src（GSSKSKPKDPSQRRRY）N端序列的两种合成肽底物具有不同的动力学参数，Km值分别为115.2 μM和44.2 μM，Vmax值分别为95和120 nmol·min-1·mg-1。表达的酶在5 mM碘乙酰胺存在下受到部分抑制（50%），在10 mM焦碳酸二乙酯存在下受到完全抑制。在酶与抑制剂的孵育中加入组氨酸可以防止后一种抑制。针对基于人源酶N端和C端序列的合成肽产生的抗血清在蛋白质印迹实验中与表达的蛋白发生反应，但只有N端序列与天然蛋白发生反应，这表明C端可能无法接近。该酶可以催化从肉豆蔻酰化肽中去除肉豆蔻酰基，但仅在添加辅酶A的情况下才会发生。
