Mutagenesis of highly conserved histidines in lecithincholesterol acyltransferase: identification of an essential histidine (His 377).
Mutagenesis of highly conserved histidines in lecithincholesterol acyltransferase: identification of an essential histidine (His 377).
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卵磷脂胆固醇酰基转移酶中高度保守的组氨酸的诱变：必需组氨酸（His 377）的鉴定。
DOI：
10.1006/bbrc.1997.7995
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Jonas,A
中科院分区：
文献类型：
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作者：
Adimoolam,S;Lee,YP;Jonas,A
卵磷脂-胆固醇酰基转移酶（LCAT）负责血浆中胆固醇酯的形成，并参与从外周组织中清除过量胆固醇。普遍认为LCAT的催化机制与丝氨酸蛋白酶和脂肪酶的催化机制类似，涉及Ser、His和酸性氨基酸残基。LCAT中的Ser 181以前被鉴定为催化残基;然而，活性位点His和酸性残基尚未被鉴定。在这项研究中，我们使用了多种方法来确定推定的活性位点组氨酸。不同物种的LCAT序列比对表明，180、263、368和377位的四个组氨酸是保守的，可能参与催化作用。基于观察到三联体的成员在大量脂肪酶的一级序列中保持相同的取向，我们排除了His 180作为潜在的候选者。突变分析沿着功能测定显示，与His 263和His 368的替换相反，用Gly、Ala或Ser替换位置377处的His消除了LCAT与界面和水溶性底物的活性，从而表明His 377在催化中的作用。
Lecithin-cholesterol acyltransferase (LCAT) is responsible for the formation of cholesterol esters in plasma and is implicated in the removal of excess cholesterol from peripheral tissues. It is generally accepted that the catalytic mechanism of LCAT is similar to that of serine proteases and lipases involving a Ser, a His, and an acidic amino acid residue. Ser181in LCAT has been previously identified as a catalytic residue; however, the active site His and acidic residue have not yet been identified. In this study we have used a variety of approaches to identify the putative active site histidine. Alignments of LCAT sequences across various species indicate that the four histidines at positions 180, 263, 368, and 377 are conserved and could be involved in catalysis. Based on the observation that the members of the triad preserve the same orientation in the primary sequence of a large number of lipases, we eliminated His180as a potential candidate. Mutational analysis along with functional assays show that, in contrast to the replacement of His263and His368, the replacement of the His at position 377 with Gly, Ala, or Ser obliterates LCAT activity with interfacial and water-soluble substrates, thus indicating a role of His377in catalysis.