Structural Basis for Bridged Bimetallic Enzyme Catalysis

描述（由申请人提供）：超过一半的蛋白质含有金属离子。其中很大一部分含有两个金属离子（通常是第一行过渡金属或镁），通过桥接配体（通常是羧酸根）连接。其中大多数是酶，它们催化各种不同的化学反应，从水解到氧化到异构化到生物聚合物合成。然而，人们对这两种金属离子如何在催化作用中一起工作，或者蛋白质环境如何调节双金属中心的内在化学反应性知之甚少。这个提议的目的是发现所有桥接的双金属酶机制的共同特征，以及蛋白质的其余部分对金属簇的化学性质的特定影响。我们选择了两种酶，链霉菌olivochromogenes木糖异构酶（XYL）和气单胞菌解蛋白氨肽酶（AAP），作为本调查的主要模式系统。木糖异构酶使用桥接的二镁中心催化糖开环，随后是醛糖-酮糖互变，这是食品工业中非常重要的反应。氨肽酶使用双锌中心水解肽和蛋白质的N-末端氨基酸;其家族成员是抗微生物、抗病毒和抗肿瘤药物的靶点。我们打算采用一系列的技术，包括定点诱变，动力学分析，抑制剂的设计和合成，超高分辨率的X射线晶体学，中子衍射，并结合量子力学/分子力学模拟，以探测AAP中的第二壳残基的作用和Xyl中的桥接配体的作用。这两种酶的晶体将X射线的分辨率提高到1 A以上，使我们能够获得极其精确的原子间参数，并确定单个原子振动和原子群集体运动的空间分布。这些数据将被用作量子力学和其他计算的输入，使我们能够看到蛋白质的其余部分如何影响双金属中心的电子分布和化学性质。我们已经有证据表明，AAP中至少一个第二壳配体的突变导致桥接二锌中心的化学性质发生显著变化：在S228 A突变体中，该酶比野生型蛋白对含硫化合物的抑制更敏感。在另一个具体目标中，我们将帮助开发一种改进蛋白质晶体结构的新方法，这种方法结合了量子力学势。此外，由于我们刚刚解决了细菌群体感应酰基高丝氨酸内酯水解酶的结构，我们将对这种具有不寻常的单齿桥的双锌酶进行类似的实验和计算。

DESCRIPTION (provided by applicant): More than half of all proteins contain metal ions. A large percentage of those contain two metal ions (usually first-row transition metals or magnesium) connected by a bridging ligand (usually a carboxylate group). Most of these are enzymes, and they catalyze a great variety of different chemical reactions, ranging from hydrolysis to oxidation to isomerization to biopolymer synthesis. Yet little is understood about how the two metal ions work together in catalysis, or how the protein environment modulates the intrinsic chemical reactivity of the dimetal center. The objective of this proposal is to discover the general features common to all bridged bimetalloenzyme mechanisms as well as the specific effects of the rest of the protein on the chemistry of the metal cluster. We have selected two enzymes, Streptomyces olivochromogenes xylose isomerase (Xyl) and Aeromonas proteolytica aminopeptidase (AAP), as primary model systems for this investigation. Xylose isomerase uses a bridged dimagnesium center to catalyze sugar ring-opening followed by aldose-ketose interconversion, a reaction of great importance in the food industry. Aminopeptidase uses a dizinc center to hydrolyze the N-terminal amino acid from peptides and proteins; members of its family are targets for antimicrobial, antiviral and antitumor drugs. We intend to employ a range of techniques, including site-directed mutagenesis, kinetic analysis, inhibitor design and synthesis, ultra-high resolution X-ray crystallography, neutron diffraction, and combined quantum mechanics/molecular mechanics simulations to probe the role of the second shell residues in AAP and the role of the bridging ligand in Xyl. Crystals of both enzymes diffract X-rays to beyond 1 A resolution, allowing us to obtain extremely precise interatomic parameters and to determine the spatial distribution of both individual atomic vibrations and collective motions of groups of atoms. These data will be used as input into quantum mechanical and other calculations, allowing us to see how the rest of the protein affects the electronic distribution and chemical properties of the dimetal center. We already have evidence that mutation of at least one of the second shell ligands in AAP causes a significant change in the chemical properties of the bridged dizinc center: in the S228A mutant, the enzyme is much more sensitive than the wild-type protein to inhibition by sulfur containing compounds. In an additional specific aim, we will help develop a new method of refining protein crystal structures, one that incorporates a quantum mechanical potential. In addition, since we have just solved the structure of a bacterial quorum-sensing acyl-homoserine lactone hydrolase, we will carry out similar experiments and calculations on this dizinc enzyme, which has an unusual monodentate bridge.