Piontek K, Chakrabarti P, Schär H P, Rossmann M G, Zuber H
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Proteins. 1990;7(1):74-92. doi: 10.1002/prot.340070108.
Structures have been determined of Bacillus stearothermophilus "apo" and holo lactate dehydrogenase. The holo-enzyme had been co-crystallized with the activator fructose 1,6-bisphosphate. The "apo" lactate dehydrogenase structure was solved by use of the known apo-M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 A to 3 A resolution by means of the noncrystallographic molecular 222 symmetry. The R-factor was reduced to 28.7%, using 2.8 A resolution data, in a restrained least-squares refinement in which the molecular symmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the "apo"-enzyme. Further refinement proceeded with the isomorphous holo-enzyme from Bacillus stearothermophilus. After removing the noncrystallographic constraints, the R-factor dropped from 30.3% to a final value of 26.0% with a 0.019 A and 1.7 degrees r.m.s. deviation from idealized bond lengths and angles, respectively. Two sulfate ions per subunit were included in the final model of the "apo"-form--one at the substrate binding site and one close to the molecular P-axis near the location of the fructose 1,6-bisphosphate activator. The final model of the holo-enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R-axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6-bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6-bisphosphate are close to the sulfate ion near the P-axis in the "apo" model. This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo-enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with nicotinamide adenine dinucleotide and oxamate than to apo-M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose 1,6-bisphosphate were also analyzed.
已确定嗜热栖热芽孢杆菌“脱辅基”和全酶乳酸脱氢酶的结构。全酶已与激活剂1,6 - 二磷酸果糖共结晶。“脱辅基”乳酸脱氢酶结构是通过使用已知的脱辅基 - M4角鲨乳酸脱氢酶分子作为起始模型来解析的。通过非晶体学分子222对称性，将相位从4埃精修并扩展至3埃分辨率。在施加分子对称性作为约束的受限最小二乘精修中，使用2.8埃分辨率数据，R因子降至28.7%。在“脱辅基”酶的四个亚基中，每个亚基均发现辅酶占有率较低。进一步使用嗜热栖热芽孢杆菌的同晶型全酶进行精修。去除非晶体学约束后，R因子从30.3%降至最终值26.0%，与理想键长和键角的均方根偏差分别为0.019埃和1.7度。“脱辅基”形式的最终模型中每个亚基包含两个硫酸根离子——一个在底物结合位点，另一个靠近1,6 - 二磷酸果糖激活剂位置附近的分子P轴。全酶的最终模型每个亚基包含两个硫酸根离子，一个在底物结合位点，另一个靠近R轴。每个亚基还包含一个烟酰胺腺嘌呤二核苷酸辅酶分子，每个四聚体包含两个1,6 - 二磷酸果糖分子。在“脱辅基”模型中，1,6 - 二磷酸果糖的磷酸基团位置靠近P轴附近的硫酸根离子。该结构代表了首次报道的变构激活乳酸脱氢酶的精修模型。激活的全酶结构与角鲨M4乳酸脱氢酶与烟酰胺腺嘌呤二核苷酸和草氨酸的三元复合物的相似性远高于与脱辅基 - M4角鲨乳酸脱氢酶的相似性。还分析了烟酰胺腺嘌呤二核苷酸和1,6 - 二磷酸果糖的构象。
