Nilsson Lill Sten O, Gao Jiali, Waldrop Grover L
Department of Chemistry and Supercomputing Institute, University of Minnesota, Minneapolis, MN 55415, USA.
J Phys Chem B. 2008 Mar 13;112(10):3149-56. doi: 10.1021/jp076326c. Epub 2008 Feb 14.
Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis in all organisms. Biotin carboxylase from Escherichia coli, whose crystal structures with and without ATP bound have been determined, has served as a model system for this component of the acetyl-CoA carboxylase complex. The two crystal structures revealed a large conformational change of one domain relative to the other domains when ATP is bound. Unfortunately, the crystal structure with ATP bound was obtained with an inactive site-directed mutant of the enzyme. As a consequence the structure with ATP bound lacked key structural information such as for the Mg2+ ions and contained altered conformations of key active-site residues. Therefore, nanosecond molecular dynamics studies of the wild-type biotin carboxylase were undertaken to supplant and amend the results of the crystal structures. Specifically, the protein-metal interactions of the two catalytically critical Mg2+ ions bound in the active site are presented along with a reevaluation of the conformations of active-site residues bound to ATP. In addition, the regions of the polypeptide chain that serve as hinges for the large conformational change were identified. The results of the hinge analysis complemented a covariance analysis that identified the individual structural elements of biotin carboxylase that change their conformation in response to ATP binding.
生物素羧化酶催化生物素的ATP依赖性羧化反应，是多酶复合物乙酰辅酶A羧化酶的一个组分，该复合物催化所有生物体脂肪酸合成中的首个关键步骤。来自大肠杆菌的生物素羧化酶，其结合和未结合ATP时的晶体结构均已确定，已成为乙酰辅酶A羧化酶复合物这一组分的模型系统。这两种晶体结构显示，当结合ATP时，一个结构域相对于其他结构域发生了较大的构象变化。不幸的是，结合ATP的晶体结构是用该酶的一个无活性的位点定向突变体获得的。因此，结合ATP的结构缺乏诸如Mg2+离子等关键结构信息，并且关键活性位点残基的构象也发生了改变。因此，对野生型生物素羧化酶进行了纳秒级分子动力学研究，以补充和修正晶体结构的结果。具体而言，展示了结合在活性位点的两个催化关键Mg2+离子的蛋白质-金属相互作用，并重新评估了与ATP结合的活性位点残基的构象。此外，确定了作为大构象变化铰链的多肽链区域。铰链分析的结果补充了协方差分析，协方差分析确定了生物素羧化酶中响应ATP结合而改变其构象的各个结构元件。
