Bucovaz E T, Macleod R M, Morrison J C, Whybrew W D
Department of Biochemistry, University of Tennessee, Memphis 38163, USA.
Biochimie. 1997 Dec;79(12):787-98. doi: 10.1016/s0300-9084(97)86938-6.
An improved procedure is described for the recovery and purification of the coenzyme A-synthesizing protein complex (CoA-SPC) of Saccharomyces cerevisiae (bakers' yeast). The molecular mass of the CoA-SPC, determined prior to and following its purification, is estimated by Sephacryl S-300 size exclusion chromatography to be between 375,000-400,000. Two previously unreported catalytic activities attributed to CoA-SPC have been identified. One of these is CoA-hydrolase activity which catalyzes the hydrolysis of CoA to form 3',5'-ADP and 4'-phosphopantetheine, and the other is dephospho-CoA-pyrophosphorylase activity which catalyzes a reaction between 4'-phosphopantetheine and ATP to form dephospho-CoA. The dephospho-CoA then reacts with ATP, catalyzed by the dephospho-CoA-kinase, to reform CoA. This sequence of reactions, referred to as the CoA/4'-phosphopantetheine cycle, provides a mechanism by which the 4'-phosphopantetheine can be recycled to form CoA. Each turn of the cycle utilizes two mol of ATP and produces one mol of ADP, one mol of PPi, and one mol of 3',5'-ADP. Other than the hydrolysis of CoA by CoA-SPC, the 4'-phosphopantetheine for the cycle apparently could be supplied by alternate sources. One alternate source may be the conventional pathway of CoA biosynthesis. Intact CoA-SPC has been separated into two segments. One segment is designated apo-CoA-SPC and the other segment segment is referred to as the 10,000-15,000 M(r) subunit. The 5'-ADP-4'-pantothenic acid-synthetase, 5'-ADP-4'-pantothenylcysteine-synthetase, 5'-ADP-4'-pantothenylcysteine-decarboxylase, and CoA-hydrolase activities reside in the apo-CoA-SPC segment of CoA-SPC. Whereas the dephospho-CoA-kinase and the dephospho-CoA-pyrophosphorylase activities reside in the 10,000-15,000 M(r) subunit. Thus, the 10,000-15,000 M(r) subunit mimics the bifunctional enzyme complex that catalyzes the final two steps in the conventional pathway of CoA biosynthesis.
本文描述了一种改进的方法，用于回收和纯化酿酒酵母（面包酵母）的辅酶A合成蛋白复合物（CoA-SPC）。通过Sephacryl S-300尺寸排阻色谱法估计，纯化前后CoA-SPC的分子量在375,000至400,000之间。已鉴定出两种先前未报道的CoA-SPC催化活性。其中一种是CoA水解酶活性，它催化CoA水解形成3',5'-ADP和4'-磷酸泛酰巯基乙胺，另一种是脱磷酸-CoA-焦磷酸化酶活性，它催化4'-磷酸泛酰巯基乙胺与ATP之间的反应形成脱磷酸-CoA。然后，脱磷酸-CoA在脱磷酸-CoA激酶的催化下与ATP反应，重新形成CoA。这一系列反应，称为CoA/4'-磷酸泛酰巯基乙胺循环，提供了一种机制，通过该机制4'-磷酸泛酰巯基乙胺可以循环形成CoA。每一轮循环消耗两摩尔ATP，产生一摩尔ADP、一摩尔焦磷酸和一摩尔3',5'-ADP。除了CoA-SPC催化CoA水解外，循环所需的4'-磷酸泛酰巯基乙胺显然可以由其他来源提供。一种替代来源可能是CoA生物合成的传统途径。完整的CoA-SPC已被分离成两个部分。一个部分称为脱辅基CoA-SPC，另一个部分称为10,000-15,000 M(r)亚基。5'-ADP-4'-泛酸合成酶、5'-ADP-4'-泛酰半胱氨酸合成酶、5'-ADP-4'-泛酰半胱氨酸脱羧酶和CoA水解酶活性存在于CoA-SPC的脱辅基CoA-SPC部分。而脱磷酸-CoA激酶和脱磷酸-CoA-焦磷酸化酶活性存在于10,000-15,000 M(r)亚基中。因此，10,000-15,000 M(r)亚基模拟了催化CoA生物合成传统途径中最后两步的双功能酶复合物。
