Haeggström J Z, Wetterholm A, Medina J F, Samuelsson B
Department of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden.
J Lipid Mediat. 1993 Mar-Apr;6(1-3):1-13.
Leukotriene (LT) A4 hydrolase (EC 3.3.2.6) is a bifunctional zinc metalloenzyme that possesses both an epoxide hydrolase activity, i.e., the well-known conversion of LTA4 into the proinflammatory substance LTB4, and a recently discovered peptidase activity. We have employed biochemical/kinetic analyses of native enzyme as well as site directed mutagenesis towards a recombinant enzyme to explore structural and functional properties of the enzyme active center. Thus, we have found that the peptidase activity is selectively stimulated by chloride ions, in a manner that suggests the presence of an anion binding site. Furthermore, a number of mutated enzymes have been constructed, expressed in E. coli, and purified to homogeneity to allow enzyme activity determinations and zinc analyses. The catalytic properties and zinc contents of these mutated enzymes establish the three zinc binding ligands of the protein and identify Glu-296 as a catalytic amino acid, directly involved in the peptidase, but not in the epoxide hydrolase reaction. In conclusion, our data provide strong evidence that the two catalytic activities of LTA4 hydrolase are exerted via non-identical but overlapping active sites.
白三烯（LT）A4水解酶（EC 3.3.2.6）是一种双功能锌金属酶，既具有环氧化物水解酶活性，即众所周知的将LTA4转化为促炎物质LTB4的过程，又具有最近发现的肽酶活性。我们采用了对天然酶的生化/动力学分析以及对重组酶的定点诱变来探索酶活性中心的结构和功能特性。因此，我们发现肽酶活性受到氯离子的选择性刺激，这种方式表明存在一个阴离子结合位点。此外，已经构建了多种突变酶，在大肠杆菌中表达并纯化至同质，以进行酶活性测定和锌分析。这些突变酶的催化特性和锌含量确定了蛋白质的三个锌结合配体，并确定Glu-296为催化氨基酸，直接参与肽酶反应，但不参与环氧化物水解酶反应。总之，我们的数据提供了强有力的证据，表明LTA4水解酶的两种催化活性是通过不相同但重叠的活性位点发挥作用的。
