Meikle P J, Whittle A M, Hopwood J J
Department of Chemical Pathology, Women's and Children's Hospital, Adelaide, Australia.
Biochem J. 1995 May 15;308 ( Pt 1)(Pt 1):327-33. doi: 10.1042/bj3080327.
Acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase) is an integral lysosomal membrane protein which catalyses the transfer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, the enzyme is capable of acetylating a number of mono- and oligo-saccharides derived from heparin, provided that a non-reducing terminal glucosamine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centrifugation and density-gradient centrifugation in Percoll. This preparation was used to investigate the kinetics of the enzyme with three acetyl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetrasaccharide derived from heparin, each containing a terminal glucosamine residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 for the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct Km values for the glucosamine substrate at both pH 7.0 and pH 5.0, whereas the tetrasaccharide substrate displayed only a single Km value at each pH. The Km values were found to be highly pH-dependent, and at pH 5.0 the values for the acetyl-acceptor substrates showed a decreasing trend as the size of the substrate increased, suggesting that the enzyme recognizes an extended region of the non-reducing terminus of the heparin or heparan sulphate polysaccharides. Double-reciprocal analysis, isotope exchange between N-acetylglucosamine and glucosamine, and inhibition studies with desulpho-CoA indicate that the enzyme operates by a random-order ternary-complex mechanism. Product inhibition studies display a complex pattern of dead-end inhibition. Taken in context with what is known about lysosomal utilization and physiological levels of acetyl-CoA, these results suggest that in vivo the enzyme operates via a random-order ternary-complex mechanism which involves the utilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.
乙酰辅酶A：α-葡糖胺N-乙酰基转移酶（N-乙酰基转移酶）是一种溶酶体膜整合蛋白，它催化乙酰基从乙酰辅酶A转移到溶酶体内肝素和硫酸乙酰肝素链末端的葡糖胺上。在体外，只要存在非还原末端葡糖胺，该酶就能使多种源自肝素的单糖和寡糖乙酰化。我们通过差速离心和在Percoll中进行密度梯度离心相结合的方法，从人胎盘中制备了高度富集的溶酶体膜组分。该制备物用于研究该酶与三种乙酰受体底物（即葡糖胺以及源自肝素的二糖和四糖，每种都含有一个末端葡糖胺残基）的动力学。该酶在pH 6.5时表现出最佳活性，对于单糖和二糖底物，活性在pH 8.0时仍能维持，但对于四糖底物，在pH 6.5以上活性急剧下降。我们在pH 7.0和pH 5.0时都确定了葡糖胺底物有两个不同的Km值，而四糖底物在每个pH值下仅显示一个Km值。发现Km值高度依赖于pH，并且在pH 5.0时，随着底物大小的增加，乙酰受体底物的值呈下降趋势，这表明该酶识别肝素或硫酸乙酰肝素多糖非还原末端的一个延伸区域。双倒数分析、N-乙酰葡糖胺和葡糖胺之间的同位素交换以及用脱硫辅酶A进行的抑制研究表明，该酶通过随机顺序的三元复合物机制起作用。产物抑制研究显示出复杂的终产物抑制模式。结合已知的溶酶体利用情况和乙酰辅酶A的生理水平，这些结果表明，在体内该酶通过随机顺序的三元复合物机制起作用，该机制涉及利用胞质乙酰辅酶A将乙酰基转移到溶酶体内肝素的末端葡糖胺残基上。
