Bame K J, Rome L H
J Biol Chem. 1985 Sep 15;260(20):11293-9.
The lysosomal membrane enzyme acetyl-CoA: alpha-glucosaminide N-acetyltransferase catalyzes the transfer of an acetyl group from acetyl-CoA to terminal alpha-linked glucosamine residues of heparan sulfate. The reaction mechanism was examined using highly purified lysosomal membranes from rat liver. The reaction was followed by measuring the acetylation of a monosaccharide acetyl acceptor, glucosamine. The enzyme reaction was optimal above pH 5.5, and a 2-3-fold stimulation of activity was observed when the membranes were assayed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicated that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. Further evidence to support this mechanism was provided by characterization of the enzyme half-reactions. Membranes incubated with acetyl-CoA and [3H]CoA were found to produce acetyl-[3H]CoA. This exchange was optimal at pH values above 7.0. Treating membranes with [3H] acetyl-CoA resulted in the formation of an acetyl-enzyme intermediate. The acetyl group could then be transferred to glucosamine, forming [3H]N-acetylglucosamine. The transfer of the acetyl group from the enzyme to glucosamine was optimal between pH 4 and 5. The results suggest that acetyl-CoA does not cross the lysosomal membrane. Instead, the enzyme is acetylated on the cytoplasmic side of the lysosome and the acetyl group is then transferred to the inside where it is used to acetylate heparan sulfate.
溶酶体膜酶乙酰辅酶A：α-葡糖胺N-乙酰基转移酶催化乙酰基从乙酰辅酶A转移至硫酸乙酰肝素末端α-连接的葡糖胺残基上。使用来自大鼠肝脏的高度纯化的溶酶体膜研究了该反应机制。通过测量单糖乙酰受体葡糖胺的乙酰化来跟踪反应。酶反应在pH 5.5以上时最佳，当在0.1%牛磺脱氧胆酸盐存在下测定膜时，观察到活性有2-3倍的刺激。双倒数分析和产物抑制研究表明该酶通过双底物有序双双乒乓机制起作用。酶半反应的表征提供了支持该机制的进一步证据。发现与乙酰辅酶A和[3H]辅酶A一起孵育的膜产生乙酰-[3H]辅酶A。这种交换在pH值高于7.0时最佳。用[3H]乙酰辅酶A处理膜导致形成乙酰化酶中间体。然后乙酰基可以转移至葡糖胺，形成[3H]N-乙酰葡糖胺。乙酰基从酶转移至葡糖胺在pH 4至5之间最佳。结果表明乙酰辅酶A不会穿过溶酶体膜。相反，该酶在溶酶体的细胞质侧被乙酰化，然后乙酰基被转移至内部，在那里用于乙酰化硫酸乙酰肝素。
