Yuan Z Y, Hammes G G
J Biol Chem. 1985 Nov 5;260(25):13532-8.
The mechanism of acyl enzyme formation from acyl-CoA derivatives was studied for chicken liver fatty acid synthase in 0.1 M potassium phosphate (pH 7.0) and 1 mM EDTA at 23 degrees C. Three mechanistically important acyl-binding sites exist: a cysteine, 4'-phosphopantetheine, and a hydroxyl (serine). The cysteine was specifically labeled with iodoacetamide, and chemical modification of this labeled enzyme with chloroacetyl-CoA resulted in additional covalent labeling of 4'-phosphopantetheine. Reaction of the enzyme with acetyl-CoA results in 47% oxyester formation, whereas with malonyl-CoA and butyryl-CoA, 57 and 80% are oxyesters, respectively, as judged by treatment of the denatured enzyme with hydroxylamine. Limited proteolysis with trypsin followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the reactive hydroxyl and cysteine are on the same peptide. Butyryl-CoA is a relatively poor primer for steady state fatty acid synthesis, probably because transfer from the hydroxyl-binding site to 4'-phosphopantetheine is inefficient. Quenched flow studies indicate that the rate constants for transfer of acetyl from enzyme-bound acetyl-CoA to native, iodoacetamide-labeled, and iodoacetamide-chloroacetyl-labeled enzyme are 43, 110, and 150 s-1. These results can be interpreted in terms of a random acylation of the hydroxyl, 4'-phosphopantetheine, and cysteine by enzyme-bound acetyl-CoA with rate constants of 150 s-1, less than 110 s-1, and less than 43 s-1, respectively. Alternatively the latter two rate constants could be characteristic of intramolecular transfer between enzyme acylation sites. Structural constraints apparently prevent all three acylation sites from being occupied simultaneously. The rate of deacetylation of the acetylated enzyme by enzyme-bound CoA also is most rapid for the iodoacetamide-chloroacetyl-labeled enzyme.
在23℃下，于0.1M磷酸钾（pH 7.0）和1mM乙二胺四乙酸（EDTA）中，研究了鸡肝脂肪酸合酶由酰基辅酶A衍生物形成酰基酶的机制。存在三个具有重要机制意义的酰基结合位点：一个半胱氨酸、4'-磷酸泛酰巯基乙胺和一个羟基（丝氨酸）。半胱氨酸用碘乙酰胺特异性标记，用氯乙酰辅酶A对该标记酶进行化学修饰导致4'-磷酸泛酰巯基乙胺的额外共价标记。用羟胺处理变性酶判断，该酶与乙酰辅酶A反应生成47%的氧酯，而与丙二酰辅酶A和丁酰辅酶A反应分别生成57%和80%的氧酯。用胰蛋白酶进行有限的蛋白水解，随后进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳表明，反应性羟基和半胱氨酸在同一肽段上。丁酰辅酶A作为稳态脂肪酸合成的引物相对较差，可能是因为从羟基结合位点转移到4'-磷酸泛酰巯基乙胺的效率较低。猝灭流动研究表明，乙酰从酶结合的乙酰辅酶A转移到天然的、碘乙酰胺标记的和碘乙酰胺-氯乙酰标记的酶的速率常数分别为43、110和150 s-1。这些结果可以解释为酶结合的乙酰辅酶A对羟基、4'-磷酸泛酰巯基乙胺和半胱氨酸的随机酰化，速率常数分别为150 s-1、小于110 s-1和小于43 s-1。或者，后两个速率常数可能是酶酰化位点之间分子内转移的特征。结构限制显然阻止了所有三个酰化位点同时被占据。酶结合的辅酶A对乙酰化酶的脱乙酰化速率对于碘乙酰胺-氯乙酰标记的酶也最快。
