Misra I, Narasimhan C, Miziorko H M
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
J Biol Chem. 1993 Jun 5;268(16):12129-35.
cDNA encoding avian liver hydroxymethylglutaryl-CoA synthase has been cloned into a pET vector, and the resulting expression plasmid has been used to transform Escherichia coli BL21 (DE3). Heterologous expression of hydroxymethylglutaryl-CoA synthase occurs upon growth of this bacterial strain in the presence of isopropyl-1-thio-beta-D-galactopyranoside, with the target enzyme representing over 20% of total cellular protein. Recombinant enzyme is soluble and stable in crude E. coli extracts, facilitating its isolation in homogeneous form. With respect to specific activity, acylation stoichiometry, Km,Ac-CoA, and binding of a spin-labeled substrate analog, the recombinant enzyme is equivalent to avian enzyme, suggesting its utility for mechanistic and structural studies. Our earlier prediction that this avian cDNA encodes the cholesterogenic cytosolic isozyme is supported by a series of experimental observations. Upon SDS-polyacrylamide gel electrophoresis, the recombinant synthase exhibits mobility in agreement with the 57.6-kDa deduced molecular mass, which exceeds the 53-kDa estimate and experimental observation for the ketogenic mitochondrial isozyme. Activity of the recombinant synthase is stimulated by Mg2+, as predicted for the cholesterogenic cytosolic isozyme and in contrast to the inhibition observed for the mitochondrial isozyme. Although antibody prepared against avian mitochondrial synthase effectively detects both avian mitochondrial and recombinant synthases on Western blots, antibody prepared against rodent cytosolic synthase discriminates between the two proteins, sensitively detecting recombinant enzyme while reacting poorly with authentic mitochondrial enzyme. Directed mutagenesis of the recombinant synthase has been performed to produce a C129S variant, in which the sulfhydryl previously implicated in formation of the acetyl-S-enzyme reaction intermediate is replaced by a hydroxyl group. EPR measurements on the binary C129S-spin-labeled acyl-CoA complex demonstrate that the mutant's substrate binding site is unperturbed in comparison with wild-type protein. These data illustrate the utility of spin-labeled substrate analogs as tools to stringently evaluate the structural integrity of engineered proteins. C129S is catalytically inactive (10(5)-fold decrease in kcat) despite retaining the ability to form noncovalent complexes with acetyl-CoA or a spin-labeled acetyl-CoA analog. The demonstrated failure of C129S to form a covalent acyl-O-enzyme species accounts for these observations; data derived from experiments performed with a C129G mutant confirm this conclusion. These results distinguish hydroxymethylglutaryl-CoA synthase from beta-ketoacyl thiolase.(ABSTRACT TRUNCATED AT 400 WORDS)
编码禽肝羟甲基戊二酰辅酶A合酶的cDNA已被克隆到pET载体中，所得表达质粒用于转化大肠杆菌BL21(DE3)。在异丙基-1-硫代-β-D-吡喃半乳糖苷存在下培养该细菌菌株时，羟甲基戊二酰辅酶A合酶会发生异源表达，目标酶占总细胞蛋白的20%以上。重组酶在大肠杆菌粗提物中可溶且稳定，便于以均一形式分离。就比活性、酰化化学计量、Km、乙酰辅酶A以及自旋标记底物类似物的结合而言，重组酶与禽酶相当，表明其可用于机理和结构研究。我们早期关于该禽cDNA编码胆固醇生成胞质同工酶的预测得到了一系列实验观察结果的支持。在SDS-聚丙烯酰胺凝胶电泳中，重组合酶的迁移率与推导的57.6 kDa分子量一致，该分子量超过了生酮线粒体同工酶的53 kDa估计值和实验观察值。重组合酶的活性受到Mg2+的刺激，这与胆固醇生成胞质同工酶的预测一致，与线粒体同工酶所观察到的抑制情况相反。尽管针对禽线粒体合酶制备的抗体在蛋白质印迹上能有效检测禽线粒体合酶和重组合酶，但针对啮齿动物胞质合酶制备的抗体能区分这两种蛋白质，能灵敏地检测重组酶，而与真正的线粒体酶反应较弱。已对重组合酶进行定向诱变以产生C129S变体，其中先前与乙酰-S-酶反应中间体形成有关的巯基被羟基取代。对二元C129S-自旋标记酰基辅酶A复合物的EPR测量表明，与野生型蛋白相比，突变体的底物结合位点未受干扰。这些数据说明了自旋标记底物类似物作为严格评估工程蛋白结构完整性工具的实用性。C129S尽管保留了与乙酰辅酶A或自旋标记的乙酰辅酶A类似物形成非共价复合物的能力，但催化无活性(kcat降低105倍)。C129S未能形成共价酰基-O-酶物种解释了这些观察结果；用C129G突变体进行实验得到的数据证实了这一结论。这些结果将羟甲基戊二酰辅酶A合酶与β-酮酰基硫解酶区分开来。(摘要截短至400字)
