Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity.
Conversion of human 15-lipoxygenase to an efficient 12-lipoxygenase: the side-chain geometry of amino acids 417 and 418 determine positional specificity.
复制标题
将人 15-脂加氧酶转化为有效的 12-脂加氧酶：氨基酸 417 和 418 的侧链几何结构决定位置特异性。
DOI：
10.1093/protein/8.3.275
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Sigal,E
中科院分区：
文献类型：
--
作者：
Sloane,DL;Leung,R;Barnett,J;Craik,CS;Sigal,E
Positional specificity determinants of human 15-lipoxygenase were examined by site-directed mutagenesis and by kinetic analysis of the wild-type and variant enzymes. By comparing conserved differences among sequences of 12- and 15-lipoxygenases, a small region responsible for functional differences between 12- and 15-lipoxygenases has been identified. Furthermore, the replacement of only two amino acids in 15-lipoxygenase (at 417 and 418 in the primary sequence) by those found in certain 12-lipoxygenases results in an enzyme that has activity similar to 12-lipoxygenase. An examination of the activity of nine variants of lipoxygenase demonstrated that the amino acid side-chain bulk and geometry of residues 417 and 418 are the key components of the positional specificity determinant of 15-lipoxygenase. Overexpression of a variant (containing valines at positions 417 and 418) that performs predominantly 12-lipoxygenation was achieved in a baculovirus-insect cell culture system. This variant was purified to >90% homogeneity and its kinetics were compared with the wild-type 15-lipoxygenase. The variant enzyme has no change in its apparentKMfor arachidonic acid and a minor(3-fold) change in itsVmax. For linoleic acid, the variant has no change in itsKMand a 10-fold reduction in itsVmax, as expected for an enzyme performing predominantly 12-lipoxygenation. The results are consistent with a model in which two amino acids of 15-lipoxygenase (isoleucine 417 and methionine 418) constitute a structural element which contributes to the regiospecificity of the enzyme. Replacement of these amino acids with those found in certain 12-lipoxygenases results in an enzyme which can bind arachidonic acid in a catalytic register that prefers 12-lipoxygenation.
