Rocque W J, McWherter C A, Wood D C, Gordon J I
Monsanto Corporate Research, Monsanto Company, St. Louis, Missouri 63198.
J Biol Chem. 1993 May 15;268(14):9964-71.
Human myristoyl-CoA:protein N-myristoyltransferase (hNmt) catalyzes the transfer of myristate from CoA to the amino-terminal Gly residue of a number of cellular proteins involved in signal transduction pathways, to structural and nonstructural proteins encoded by retroviruses, hepadnaviruses, picornaviruses, and reoviruses, as well as to several transforming tyrosine kinases. hNmt has been purified 230-fold from an erythroleukemia cell line. The monomeric enzyme has no associated methionyl aminopeptidase activity. To determine the enzyme's kinetic mechanism, we examined the effect of covariation of subsaturating concentrations of myristoyl-CoA and peptide substrate on initial velocity. Double-reciprocal plots excluded a double displacement (ping-pong) mechanism. Product inhibition studies indicated that CoA was a noncompetitive inhibitor against myristoyl-CoA and a mixed-type inhibitor against peptide substrates. Together these results are consistent with a sequential ordered mechanism where, in a typical catalytic cycle, myristoyl-CoA binds to apoenzyme before peptide followed by release of the CoA and then myristoylpeptide products. This kinetic mechanism is identical to that described for Saccharomyces cerevisiae N-myristoyl-transferase (Nmt1p) and emphasizes the impact that regulation of myristoyl-CoA pool size and accessibility may have in modulating protein N-myristoylation in these two species. Comparative studies of the peptide substrate specificities of hNmt and Nmt1p using a panel of 12 octapeptides revealed distinct differences in their tolerance for amino acid substitutions at positions 3, 4, 7, and 8 of parental peptides derived from the amino-terminal sequences of known N-myristoyl-proteins. This finding contrasts with our recent observation that the acyl-CoA substrate specificities of hNmt and Nmt1p are highly conserved and suggests that these differences in peptide recognition provide an opportunity to develop species-specific enzyme inhibitors.
人豆蔻酰辅酶A：蛋白质N-豆蔻酰基转移酶（hNmt）催化豆蔻酸从辅酶A转移至参与信号转导途径的多种细胞蛋白、逆转录病毒、嗜肝DNA病毒、小RNA病毒和呼肠孤病毒编码的结构及非结构蛋白以及几种转化型酪氨酸激酶的氨基端甘氨酸残基上。hNmt已从一种红白血病细胞系中纯化了230倍。该单体酶不具有相关的甲硫氨酰氨基肽酶活性。为确定该酶的动力学机制，我们研究了亚饱和浓度的豆蔻酰辅酶A和肽底物的协变对初速度的影响。双倒数作图排除了双置换（乒乓）机制。产物抑制研究表明，辅酶A是针对豆蔻酰辅酶A的非竞争性抑制剂，是针对肽底物的混合型抑制剂。这些结果共同表明其符合顺序有序机制，即在典型的催化循环中，豆蔻酰辅酶A在肽之前结合至脱辅酶，随后释放辅酶A，然后是豆蔻酰肽产物。这种动力学机制与酿酒酵母N-豆蔻酰转移酶（Nmt1p）所描述的相同，并强调了豆蔻酰辅酶A库大小和可及性的调节在调节这两个物种中蛋白质N-豆蔻酰化方面可能产生的影响。使用一组12种八肽对hNmt和Nmt1p的肽底物特异性进行的比较研究显示，它们对源自已知N-豆蔻酰化蛋白氨基端序列的亲本肽第3、4、7和8位氨基酸取代的耐受性存在明显差异。这一发现与我们最近观察到的hNmt和Nmt1p的酰基辅酶A底物特异性高度保守形成对比，并表明这些肽识别方面的差异为开发物种特异性酶抑制剂提供了机会。
