Sloane D L, Leung R, Barnett J, Craik C S, Sigal E
Syntex Discovery Research, Palo Alto, CA 94303, USA.
Protein Eng. 1995 Mar;8(3):275-82. doi: 10.1093/protein/8.3.275.
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 baculo-virus-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 apparent KM for arachidonic acid and a minor (3-fold) change in its Vmax. For linoleic acid, the variant has no change in its KM and a 10-fold reduction in its Vmax, 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.
通过定点诱变以及对野生型和变体酶的动力学分析，研究了人15-脂氧合酶的位置特异性决定因素。通过比较12-脂氧合酶和15-脂氧合酶序列之间的保守差异，已确定了一个导致12-脂氧合酶和15-脂氧合酶功能差异的小区域。此外，在15-脂氧合酶中（一级序列中的第417和418位）仅用某些12-脂氧合酶中发现的两个氨基酸进行替换，会产生一种活性类似于12-脂氧合酶的酶。对九种脂氧合酶变体活性的研究表明，第417和418位残基的氨基酸侧链体积和几何形状是15-脂氧合酶位置特异性决定因素的关键组成部分。在杆状病毒-昆虫细胞培养系统中实现了主要进行12-脂氧合作用的变体（第417和418位为缬氨酸）的过表达。该变体被纯化至>90%的纯度，并将其动力学与野生型15-脂氧合酶进行了比较。变体酶对花生四烯酸的表观KM没有变化，Vmax有轻微（3倍）变化。对于亚油酸，该变体的KM没有变化，Vmax降低了10倍，这与主要进行12-脂氧合作用的酶预期的情况一致。结果与一个模型相符，即15-脂氧合酶的两个氨基酸（异亮氨酸417和甲硫氨酸418）构成一个结构元件，该元件有助于酶的区域特异性。用某些12-脂氧合酶中发现的氨基酸替换这些氨基酸会产生一种酶，该酶能够以更倾向于12-脂氧合作用的催化配准方式结合花生四烯酸。
