Cloning, heterologous expression, and enzymological characterization of human squalene monooxygenase
Cloning, heterologous expression, and enzymological characterization of human squalene monooxygenase
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DOI：
10.1006/abbi.1999.1629
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发表时间：
2000-02-15
影响因子：
3.9
通讯作者：
Porter, TD
中科院分区：
文献类型：
--
作者：
Laden, BP;Tang, YZ;Porter, TD
人角鲨烯单加氧酶是胆固醇生物合成关键途径中的一种关键酶，其cDNA从人肝脏cDNA文库中扩增并克隆，该蛋白在大肠杆菌中表达并纯化。对纯化酶的动力学分析显示，角鲨烯的表观Km为7.7μM，表观kcat为1.1 min⁻¹。对于FAD，表观Km为0.3μM，这与松散结合的黄素相符。对于NADPH - 细胞色素P450还原酶（必需的电子传递伙伴），表观Km为14 nM。达到最大活性所需的还原酶量比测定中存在的角鲨烯单加氧酶量少约三倍；因此，向单加氧酶的电子传递不太可能是限速步骤。先前的报道暗示该酶的抑制是喂食含碲饮食的断奶大鼠出现外周脱髓鞘的原因。由于没有人类的数据，研究了多种碲及相关元素化合物抑制重组人酶的能力。亚碲酸盐、二氧化碲、亚硒酸盐和二氧化硒具有抑制作用；碲化合物比硒化合物更有效，从它们的IC₅₀值（分别为17和37μM）可以看出。亚碲酸盐抑制的动力学分析表明，相对于角鲨烯，它与酶以非竞争性方式在多个位点相互作用。（C）2000学术出版社
The cDNA for human squalene monooxygenase, a key enzyme in the committed pathway for cholesterol biosynthesis, was amplified from a human liver cDNA library and cloned, and the protein was expressed in Escherichia coli and purified. Kinetic analysis of the purified enzyme revealed an apparent K-m for squalene of 7.7 mu M and an apparent k(cat) of 1.1 min-1. For FAD the apparent K-m is 0.3 mu M, consistent with a loosely bound flavin. The apparent K-m for NADPH-cytochrome P450 reductase, the requisite electron transfer partner, is 14 nM. The amount of reductase needed for maximal activity is about threefold less than the amount of squalene monooxygenase present in the assay; thus, electron transfer to the monooxygenase is not likely to be rate limiting. Previous reports have implicated inhibition of this enzyme as the cause of a peripheral demyelination seen in weanling rats fed a diet containing tellurium. As no data were available for humans, the ability of a number of tellurium and related elemental compounds to inhibit the recombinant human enzyme was examined. Tellurite, tellurium dioxide, selenite, and selenium dioxide were inhibitory; the tellurium compounds were more potent than the selenium compounds, as indicated by their IC50 values (17 and 37 mu M, respectively). Kinetic analysis of the inhibition by tellurite suggests multiple sites of interaction with the enzyme in a noncompetitive manner with respect to squalene. (C) 2000 Academic Press.