Pederson B A, Cheng C, Wilson W A, Roach P J
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, and the Indiana University Center for Diabetes Research, Indianapolis, Indiana 46202-5122, USA.
J Biol Chem. 2000 Sep 8;275(36):27753-61. doi: 10.1074/jbc.M003342200.
The major yeast glycogen synthase, Gsy2p, is inactivated by phosphorylation and activated by the allosteric ligand glucose-6-P. From studies of recombinant proteins, the control can be accommodated by a three-state model, in which unphosphorylated enzyme has intermediate activity (state II). Glucose-6-P increased V(max)/K(m) by about 2-fold (state III), whereas phosphorylation by the cyclin-dependent protein kinase Pcl10p/Pho85p decreased V(max)/K(m) by approximately 30-fold (state I). In the presence of glucose-6-P, state III is achieved regardless of phosphorylation state. The enzyme forms complexes in solution with the yeast glycogenin Glg2p, but this interaction appears not to affect control either by glucose-6-P binding or by phosphorylation. Scanning mutagenesis was applied to identify residues potentially involved in ligand binding. Of 22 mutant enzymes analyzed, seven were essentially inactive. Five mutant proteins were altered in their activation by glucose-6-P, and two were completely unaffected by the hexose phosphate. One of these, R586A/R588A/R591A (all three of the indicated Arg residues mutated to Ala), had wild-type activity and was normally inactivated by phosphorylation. A second mutant, R579A/R580A/R582A, had somewhat reduced V(max), but its activity was not greatly reduced by phosphorylation. The Arg residues in these two mutants are restricted to a highly conserved, 13-residue segment of Gsy2p that we propose to be important for glucose-6-P binding and/or the ability of the enzyme to undergo transitions between activity states.
主要的酵母糖原合酶Gsy2p通过磷酸化而失活，并被变构配体葡萄糖-6-磷酸激活。通过对重组蛋白的研究，这种调控可以用三态模型来解释，其中未磷酸化的酶具有中等活性（状态II）。葡萄糖-6-磷酸使V(max)/K(m)增加约2倍（状态III），而细胞周期蛋白依赖性蛋白激酶Pcl10p/Pho85p的磷酸化使V(max)/K(m)降低约30倍（状态I）。在存在葡萄糖-6-磷酸的情况下，无论磷酸化状态如何都能达到状态III。该酶在溶液中与酵母糖原素Glg2p形成复合物，但这种相互作用似乎不影响葡萄糖-6-磷酸结合或磷酸化对其的调控。应用扫描诱变来鉴定可能参与配体结合的残基。在分析的22种突变酶中，有7种基本无活性。5种突变蛋白被葡萄糖-6-磷酸激活的情况发生了改变，2种完全不受己糖磷酸的影响。其中一种，R586A/R588A/R591A（所有三个指定的精氨酸残基都突变为丙氨酸），具有野生型活性，并且通常通过磷酸化失活。第二个突变体，R579A/R580A/R582A，其V(max)有所降低，但其活性并未因磷酸化而大幅降低。这两个突变体中的精氨酸残基局限于Gsy2p高度保守的13个残基片段，我们认为该片段对于葡萄糖-6-磷酸结合和/或酶在活性状态之间转变的能力很重要。
