Rajasekharan R, Marians R C, Shockey J M, Kemp J D
Plant Genetic Engineering Laboratory, New Mexico State University, Las Cruces 88003.
Biochemistry. 1993 Nov 23;32(46):12386-91. doi: 10.1021/bi00097a016.
Synthesis of 32P-labeled CoA of high specific activity was achieved using partially purified dephospho-CoA kinase (EC 2.7.1.24) from pig liver with [gamma-32P]ATP as donor and dephospho-CoA as acceptor. A photoaffinity dodecanoic acid analog, 12-[(4-azidosalicyl)amino]dodecanoic acid was synthesized, as were its CoA derivative (ASD-CoA) and the CoA derivative of 12-azidooleic acid. The CoA derivatives were synthesized from azido fatty acid analogs by acyl-CoA synthetase. The synthesized photolabile reagents were tested as photoaffinity labels for acyl-CoA oxidase (EC 1.3.99.3) from Arthrobacter species. When a mixture of oxidase and the acyl-CoA analogs were incubated in the absence of ultraviolet light, the analogs were recognized as substrate. Acyl-CoA oxidase was incubated in the presence of acyl-CoA analogs and immediately photolyzed, which resulted in irreversible inhibition. Oleoyl-CoA and dodecanoyl-CoA protect the enzyme from photoactivated inhibition by 12-azidooleoyl-CoA and ASD-CoA, respectively. Analysis of photolyzed enzyme preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that both analogs preferentially labeled a 54,000 molecular weight protein. These results demonstrate that the photoaffinity acyl-CoA analogs have potential application as probes to identify and characterize lipid biosynthetic enzymes and to identify the active site of these proteins.
使用猪肝中部分纯化的脱磷酸辅酶A激酶（EC 2.7.1.24），以[γ-32P]ATP作为供体、脱磷酸辅酶A作为受体，成功合成了高比活性的32P标记辅酶A。合成了一种光亲和十二烷酸类似物12-[(4-叠氮基水杨基)氨基]十二烷酸，以及它的辅酶A衍生物（ASD-CoA）和12-叠氮基油酸的辅酶A衍生物。这些辅酶A衍生物由叠氮脂肪酸类似物通过酰基辅酶A合成酶合成。所合成的光不稳定试剂被用作节杆菌属酰基辅酶A氧化酶（EC 1.3.99.3）的光亲和标记物进行测试。当氧化酶与酰基辅酶A类似物的混合物在无紫外光条件下孵育时，这些类似物被识别为底物。酰基辅酶A氧化酶在酰基辅酶A类似物存在下孵育并立即进行光解，导致不可逆抑制。油酰辅酶A和十二烷酰辅酶A分别保护该酶免受12-叠氮基油酰辅酶A和ASD-CoA的光活化抑制。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和放射自显影对光解的酶制剂进行分析，结果显示这两种类似物都优先标记了一种分子量为54,000的蛋白质。这些结果表明，光亲和酰基辅酶A类似物作为探针在鉴定和表征脂质生物合成酶以及确定这些蛋白质的活性位点方面具有潜在应用价值。
