HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.
HlyC, the internal protein acyltransferase that activates hemolysin toxin: the role of conserved tyrosine and arginine residues in enzymatic activity as probed by chemical modification and site-directed mutagenesis.
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HlyC，激活溶血素毒素的内部蛋白酰基转移酶：通过化学修饰和定点诱变探测保守酪氨酸和精氨酸残基在酶活性中的作用。
DOI：
10.1021/bi990138y
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发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Ernst-Fonberg,ML
中科院分区：
文献类型：
--
作者：
Trent,MS;Worsham,LM;Ernst-Fonberg,ML
蛋白质的内部脂肪酰化是一种公认的改变生物学行为的手段。大肠杆菌溶血素A（HlyA）是一种毒性蛋白质，通过两个赖氨酸残基的ε-氨基的内部酰化而被转录为无毒蛋白质并具有毒性; HlyC催化酰基−酰基载体蛋白（ACP）的酰基转移，ACP是专性酰基供体。激活13种不同RTX（毒素中的重复序列）毒素（HlyA是其中的原型）的相应同源C蛋白中的保守残基可能包括一些在催化中重要的残基。两个保守的酪氨酸和两个保守的酪氨酸的可能的作用进行了研究，注意到化学修饰剂和定点突变的影响。在pH 8.0下HlyC的TNM修饰导致广泛的抑制，该抑制被底物肉豆蔻酰-ACP的存在阻止，但不被产物ACPSH阻止。NAI没有效果。Y 70 G和Y150 G大大降低了酶活性，而突变Y 70 F和Y150 F表现出野生型活性。修饰精氨酸残基与PG显着降低酰基转移酶活性与肉豆蔻酰-ACP和ACPSH的中度保护。在最佳条件下，两个保守的精氨酸残基（R24 A，R24 K，R87 A和R87 K）的四个单独的突变对酰基转移酶活性几乎没有影响。
Internal fatty acylation of proteins is a recognized means of modifying biological behavior.Escherichia colihemolysin A (HlyA), a toxic protein, is transcribed as a nontoxic protein and made toxic by internal acylation of two lysine residue ε-amino groups; HlyC catalyzes the acyl transfer from acyl−acyl carrier protein (ACP), the obligate acyl donor. Conserved residues among the respective homologous C proteins that activate 13 different RTX (repeats in toxin) toxins of which HlyA is the prototype likely include some residues that are important in catalysis. Possible roles of two conserved tyrosines and two conserved arginines were investigated by noting the effects of chemical modifiers and site-directed mutagenesis. TNM modification of HlyC at pH 8.0 led to extensive inhibition that was prevented by the presence of the substrate myristoyl-ACP but not by the product, ACPSH. NAI had no effect. Y70G and Y150G greatly diminished enzyme activity, whereas mutations Y70F and Y150F exhibited wild-type activity. Modification of arginine residues with PG markedly lowered acyltransferase activity with moderate protection by both myristoyl-ACP and ACPSH. Under optimum conditions, four separate mutations of the two conserved arginine residues (R24A, R24K, R87A, and R87K) had little effect on acyltransferase activity.