Coats W S, Browner M F, Fletterick R J, Newgard C B
Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1991 Aug 25;266(24):16113-9.
Liver and muscle glycogen phosphorylases, which are products of distinct genes, are both activated by covalent phosphorylation, but in the unphosphorylated (b) state, only the muscle isozyme is efficiently activated by the allosteric activator AMP. The different responsiveness of the phosphorylase isozymes to allosteric ligands is important for the maintenance of tissue and whole body glucose homeostasis. In an attempt to understand the structural determinants of differential sensitivity of the muscle and liver isozymes to AMP, we have developed a bacterial expression system for the liver enzyme, allowing native and engineered proteins to be expressed and characterized. Engineering of the single amino acid substitutions Thr48Pro, Met197Thr and the double mutant Thr48Pro, Met197Thr in liver phosphorylase, and Pro48Thr in muscle phosphorylase, did not qualitatively change the response of the two isozymes to AMP. These sites had previously been implicated in the configuration of the AMP binding site. However, when nine amino acids among the first 48 in liver phosphorylase were replaced with the corresponding muscle phosphorylase residues (L1M2-48L49-846), the engineered liver enzyme was activated by AMP to a higher maximal activity than native liver phosphorylase. Interestingly, the homotropic cooperativity of AMP binding was unchanged in the engineered phosphorylase b protein, and heterotropic cooperativity between the glucose-1-phosphate and AMP sites was only slightly enhanced. The native liver, native muscle and L1M2-48L49-846 phosphorylases were converted to the a form by treatment with purified phosphorylase kinase; the maximal activity of the chimeric a enzyme was greater than the native liver a enzyme and approached that of muscle phosphorylase a. From these results we suggest that tissue-specific phosphorylase isozymes have evolved a complex mechanism in which the N-terminal 48 amino acids modulate intrinsic activity (Vmax), probably by affecting subunit interactions, and other, as yet undefined regions specify the allosteric interactions with ligands and substrates.
肝脏和肌肉糖原磷酸化酶是不同基因的产物，二者均通过共价磷酸化被激活，但在未磷酸化（b）状态下，只有肌肉同工酶能被变构激活剂AMP有效激活。磷酸化酶同工酶对变构配体的不同反应性对于维持组织和全身葡萄糖稳态很重要。为了理解肌肉和肝脏同工酶对AMP敏感性差异的结构决定因素，我们开发了一种用于肝脏酶的细菌表达系统，可表达和表征天然及工程化蛋白质。对肝脏磷酸化酶中的单氨基酸取代Thr48Pro、Met197Thr以及双突变体Thr48Pro、Met197Thr，还有肌肉磷酸化酶中的Pro48Thr进行工程改造，并未定性改变两种同工酶对AMP的反应。这些位点先前被认为与AMP结合位点的构象有关。然而，当将肝脏磷酸化酶前48个氨基酸中的9个替换为相应的肌肉磷酸化酶残基（L1M2 - 48L49 - 846）时，工程化肝脏酶被AMP激活至比天然肝脏磷酸化酶更高的最大活性。有趣的是，在工程化的磷酸化酶b蛋白中，AMP结合的同向协同性未变，而葡萄糖 - 1 - 磷酸和AMP位点之间的异向协同性仅略有增强。通过用纯化的磷酸化酶激酶处理，将天然肝脏、天然肌肉和L1M2 - 48L49 - 846磷酸化酶转化为a形式；嵌合a酶的最大活性大于天然肝脏a酶，接近肌肉磷酸化酶a的活性。从这些结果我们推测，组织特异性磷酸化酶同工酶已进化出一种复杂机制，其中N端48个氨基酸可能通过影响亚基相互作用来调节内在活性（Vmax），而其他尚未明确的区域则决定与配体和底物的变构相互作用。
