Miller S J, Parker R A, Gibson D M
Department of Biochemistry, Indiana University School of Medicine, Indianapolis 46223.
Adv Enzyme Regul. 1989;28:65-77. doi: 10.1016/0065-2571(89)90064-2.
3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is the limiting enzyme step in cholesterol formation in mammalian liver and other tissues. It is a glycoprotein of 97,000 daltons embedded in the endoplasmic reticulum with a long cytoplasmic extension that is the site of catalytic conversion of HMG CoA to mevalonate. The enzyme is subject to both long-term (induction/repression; degradation) and short-term control (reversible phosphorylation) mediated by endocrine signaling (insulin, glucagon) and through negative feedback by metabolic products of mevalonate (e.g., cholesterol). The catalytic capacity of microsomal reductase falls rapidly in the presence of several protein kinases (reductase kinase, protein kinase-C, calmodulin-dependent protein kinase). Activity is restored with various protein phosphatases. Increased phosphorylation of reductase in intact cells after addition of glucagon or mevalonate is followed by enhanced degradation of the enzyme. In an in vitro model system, phosphorylated, native microsomal reductase is more rapidly cleaved by the calcium-dependent, neutral protease calpain than the dephosphorylated from of reductase. Our present research which centers on the mechanism of the in vitro model system is reviewed. Calpain in the presence of Ca2+ cleaves the cytosolic domain of phosphorylated 97 kDa reductase at two points giving rise to two fragments of nearly the same size that appear as a 52-56,000 dalton doublet by electrophoresis and immunoblotting. In the same system native reductase labeled with [gamma-32P]ATP generates a doublet with 32P solely in the upper (heavier) band. This indicates that serine phosphorylation sites lie between the two calpain cleavage loci. These are positioned in the "linker" region of the long carboxy-terminal cytosolic domain near the membrane. This segment possesses five invariant serine residues and two PEST sequences (constellations of proline, glutamate, serine and threonine) that are characteristic of proteins with short half-lives. If phosphorylation of HMG CoA reductase is confined to the linker region, we must look to this domain in order to interpret the resulting conformational changes that markedly influence reductase catalytic activity and prepare the enzyme for degradation.
3-羟基-3-甲基戊二酰辅酶A（HMG CoA）还原酶是哺乳动物肝脏和其他组织中胆固醇合成的限速酶。它是一种97000道尔顿的糖蛋白，嵌入内质网，具有长的胞质延伸部分，是HMG CoA催化转化为甲羟戊酸的部位。该酶受到由内分泌信号（胰岛素、胰高血糖素）介导的长期（诱导/抑制；降解）和短期控制（可逆磷酸化），以及甲羟戊酸代谢产物（如胆固醇）的负反馈调节。在几种蛋白激酶（还原酶激酶、蛋白激酶C、钙调蛋白依赖性蛋白激酶）存在的情况下，微粒体还原酶的催化能力迅速下降。各种蛋白磷酸酶可恢复其活性。添加胰高血糖素或甲羟戊酸后，完整细胞中还原酶的磷酸化增加，随后该酶的降解增强。在体外模型系统中，磷酸化的天然微粒体还原酶比去磷酸化的还原酶更易被钙依赖性中性蛋白酶钙蛋白酶快速切割。本文综述了我们目前以体外模型系统机制为中心的研究。在Ca2+存在的情况下，钙蛋白酶在两个位点切割磷酸化的97 kDa还原酶的胞质结构域，产生两个大小几乎相同的片段，通过电泳和免疫印迹显示为52000-56000道尔顿的双峰。在同一系统中，用[γ-32P]ATP标记的天然还原酶仅在上部（较重）条带中产生含32P的双峰。这表明丝氨酸磷酸化位点位于两个钙蛋白酶切割位点之间。这些位点位于靠近膜的长羧基末端胞质结构域的“连接区”。该片段具有五个不变的丝氨酸残基和两个PEST序列（脯氨酸、谷氨酸、丝氨酸和苏氨酸的组合），这是半衰期短的蛋白质的特征。如果HMG CoA还原酶的磷酸化局限于连接区，我们必须关注该结构域，以便解释由此产生的显著影响还原酶催化活性并使酶准备降解的构象变化。
