Activation of hemolysin toxin: relationship between two internal protein sites of acylation.
Activation of hemolysin toxin: relationship between two internal protein sites of acylation.
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溶血素毒素的激活：两个内部蛋白质酰化位点之间的关系。
DOI：
10.1021/bi035919k
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Ernst-Fonberg,MLou
中科院分区：
文献类型：
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作者：
Langston,KeishaG;Worsham,LesaMS;Earls,Laurie;Ernst-Fonberg,MLou
HlyC是溶血素激活的赖氨酸酰基转移酶，它催化大肠杆菌原溶血素(ProHlyA)在特定赖氨酸残基的ε-氨基上发生酰化反应，形成溶血素。两个酰化位点两侧的氨基酸序列不是同源的，除了每个氨基酸都有一个甘氨酸残基紧接在被酰化的赖氨酸之前；然而，在整个proHlyA中有许多GK序列不是酰化位点。考察了酰化反应的底物专一性。ProHlyA衍生的结构通常作为蛋白质内部的酰化底物，通过大幅删除和分离酰化位点而改变为两个不同的肽，并对其进行定点突变分析，并测量了酰化反应的动力学。ProHlyA的两个酰化位点与HlyC相互独立地发挥作用；这两个位点之间似乎没有共同的HlyC结合位点或酶的可加工性。酰基-HlyC可能是与最终的酰化底物相互作用的酶形式。在不同的结构中，两个酰化位点具有相似的Km值，但它们作为底物的Vmax值和催化效率不同。除了关键的赖氨酸被精氨酸取代外，模拟每个酰化位点的一级结构的特定小肽可以抑制蛋白质的内部酰化；然而，含有关键赖氨酸的类似小肽不被酰化。
HlyC, hemolysin-activating lysine acyltransferase, catalyzes the acylation (from acyl-ACP) ofEscherichia coliprohemolysin (proHlyA) on the ε-amino groups of specific lysine residues, Lys564 and Lys690 of the 1024-amino acid primary structure, to form hemolysin (HlyA). The amino acid sequences flanking the two acylation sites are not homologous except that each has a glycine residue immediately preceding the lysine which is acylated; there are, however, numerous GK sequences throughout proHlyA that are not acylation sites. The substrate specificity of acylation was examined. ProHlyA-derived structures, altered by substantial deletions and separation of the acylation sites into two different peptides and site-directed mutation analyses of acylation sites, often served as internal protein acylation substrates, and the kinetics of the acylations were measured. The two sites of acylation of proHlyA functioned independently of one another with HlyC; there did not appear to be a common HlyC binding site or processivity of the enzyme between the sites. Acyl-HlyC was likely the enzyme form that interacted with the final acylation substrate. In a variety of constructs, the two acylation sites had similarKmvalues, but theirVmaxvalues and catalytic efficiencies as substrates differed. Internal protein acylation was inhibited by specific small peptides mimicking the primary structure of each acylation site except that the crucial lysines were replaced with arginines; similar small peptides containing the crucial lysine, however, were not acylated.