Um H D, Klein C
E.A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, MO 63104.
Biochem J. 1993 Nov 1;295 ( Pt 3)(Pt 3):821-6. doi: 10.1042/bj2950821.
We have previously reported that distinctly different concentrations of GDP stimulate the phosphorylation and dephosphorylation of p36, the alpha-subunit of succinyl-CoA synthetase (SCS) in Dictyostelium discoideum. In this present study, we have investigated the mechanism underlying these dual effects of GDP. Dephosphorylation of p36 is induced by relatively high levels of GDP and is coincident with the formation of GTP. This indicates that, at high concentrations, GDP serves as a substrate of SCS. However, 100-fold lower concentrations of GDP, which do not bind to the catalytic site to induce SCS dephosphorylation, stimulate p36 phosphorylation. This stimulation is not diminished by dilution of the sample, and is retained during purification of the protein. Gel-filtration analyses indicate that SCS in our system behaves as a non-interacting alpha beta dimer, the hydrodynamic behaviour of which is not altered by the presence of added GDP. The data indicate that altered protein-protein interactions do not account for the stimulation of p36 phosphorylation by low GDP concentrations. We propose that GDP functions as an allosteric regulator of SCS, and experiments using guanosine 5'-[beta-thio]diphosphate (GDP[S]) are shown to distinguish further the allosteric and catalytic binding sites.
我们之前报道过，不同浓度的GDP能刺激盘基网柄菌中琥珀酰辅酶A合成酶（SCS）的α亚基p36的磷酸化和去磷酸化。在本研究中，我们探究了GDP产生这些双重作用的机制。p36的去磷酸化由相对高水平的GDP诱导，且与GTP的形成同时发生。这表明，在高浓度时，GDP作为SCS的底物。然而，浓度低100倍的GDP不与催化位点结合以诱导SCS去磷酸化，却能刺激p36磷酸化。这种刺激不会因样品稀释而减弱，且在蛋白质纯化过程中保持。凝胶过滤分析表明，我们系统中的SCS表现为非相互作用的αβ二聚体，添加的GDP的存在不会改变其流体动力学行为。数据表明，蛋白质 - 蛋白质相互作用的改变不能解释低浓度GDP对p36磷酸化的刺激作用。我们提出GDP作为SCS的变构调节剂起作用，并且使用鸟苷5'-[β-硫代]二磷酸（GDP[S]）的实验进一步区分了变构结合位点和催化结合位点。
