Park Young-Ha, Lee Byeong R, Seok Yeong-Jae, Peterkofsky Alan
Department of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul 151-742, Korea.
J Biol Chem. 2006 Mar 10;281(10):6448-54. doi: 10.1074/jbc.M512672200. Epub 2006 Jan 9.
A widely accepted model for catabolite repression posits that phospho-IIAGlc of the bacterial phosphotransferase system activates adenylyl cyclase (AC) activity. For many years, attempts to observe such regulatory properties of AC in vitro have been unsuccessful. To further study the regulation, AC was produced fused to the transmembrane segments of the serine chemoreceptor Tsr. Cells harboring Tsr-AC and normal AC, expressed from the cya promoter on a low copy number vector, exhibit similar behavior with respect to elevation of cAMP levels resulting from deletion of crp, expressing the catabolite regulatory protein. Membrane-bound Tsr-AC exhibits activity comparable with the native form of AC. Tsr-AC binds IIAGlc specifically, regardless of its phosphorylation state, but not the two general phosphotransferase system proteins, enzyme I and HPr; IIAGlc binding is localized to the C-terminal region of AC. Binding to membranes of either dephospho- or phospho-IIAGlc has no effect on AC activity. However, in the presence of an Escherichia coli extract, P-IIAGlc, but not IIAGlc, stimulates AC activity. Based on these findings of a direct interaction of IIAGlc with AC, but activity regulation only in the presence of E. coli extract, a revised model for AC activity regulation is proposed.
一种被广泛接受的分解代谢物阻遏模型认为，细菌磷酸转移酶系统的磷酸化IIAGlc会激活腺苷酸环化酶（AC）的活性。多年来，在体外观察AC这种调节特性的尝试均未成功。为了进一步研究这种调节作用，将AC与丝氨酸化学感受器Tsr的跨膜片段融合表达。携带Tsr-AC和正常AC的细胞（由低拷贝数载体上的cya启动子表达），在因缺失表达分解代谢调节蛋白的crp而导致cAMP水平升高方面表现出相似的行为。膜结合的Tsr-AC表现出与天然形式的AC相当的活性。Tsr-AC特异性结合IIAGlc，无论其磷酸化状态如何，但不结合磷酸转移酶系统的两种通用蛋白，即酶I和HPr；IIAGlc的结合定位于AC的C末端区域。与去磷酸化或磷酸化的IIAGlc的膜结合对AC活性没有影响。然而，在存在大肠杆菌提取物的情况下，磷酸化IIAGlc（P-IIAGlc）而非IIAGlc能刺激AC活性。基于IIAGlc与AC直接相互作用但仅在存在大肠杆菌提取物时才进行活性调节的这些发现，提出了一种AC活性调节的修订模型。
