CATALYTIC ANTIBODY DESIGN & CHARACTERIZATION

在一个旨在了解基本结构-功能的项目中， 在酶催化的关系，我们已经创建了一个新的家庭， 来自免疫球蛋白分子的酶样催化剂。 我们两个最 活性催化抗体17 E8和29 G11，通过以下方法制备： 用正亮氨酸苯基膦酸酯过渡态免疫 类似物，并催化正亮氨酸苯酯的水解 印刷受体. 这些催化抗体相当有效 酯酶。 初步的机理实验表明，17 E8 酯解通过形成共价酰基酶进行 类似于丝氨酸蛋白酶的中间体，而29 G11 酯水解包括水直接攻击底物羰基 组 在与罗伯特·弗莱彻的团队合作中， 解析了17 E8 -1复合物的晶体结构。 结构 揭示了17 E8的活性位点与 天然三联体水解酶如丝氨酸 蛋白酶和乙酰胆碱酯酶。 17 E8活性位点含有 Ser-His催化二联体代替Ser-His-Asp催化三联体， 天然水解酶 此外，17 E8活性位点具有 质子化的赖氨酸侧链，我们认为它的功能是“含氧阴离子 孔”相当于稳定氧阴离子字符的发展， 水解过渡态。 相反，天然水解酶 通过氢键相互作用调节含氧阴离子稳定化， 主链酰胺NH基团。 这些数据表明， 与涉及亲核攻击的天然酶类似的机制 我们目前正在测试这一假设， 机械探针，定点诱变，和进一步的结构 特征化

In a project aimed at understanding basic structure-function relations in enzymatic catalysis, we have created a new family of enzyme-like catalysts from an immunoglobulin molecules. Our two most active catalytic antibodies, 17E8 and 29G11, prepared through immunization with a norleucine phenyl phosphonate transition state analog, and catalyze the hydrolysis norleucine phenyl ester substrates. These catalytic antibodies are fairly efficient esterases. Preliminary mechanistic experiments suggest that 17E8 esterolysis proceeds through the formation of a covalent acyl-enzyme intermediate in analogy to the serine proteases, whereas 29G11 esterolysis involves direct attack of water at the substrate carbonyl group. In a collaboration with Robert Fletterick's group we have solved the crystal structure of the 17E8-1 complex. The structure reveals a remarkable similarity between the active site of 17E8 and active sites of natural triad-based hydrolases such as the serine proteases and acetyl cholinesterase. The 17E8 active site contains a Ser-His catalytic dyad instead of the Ser-His-Asp catalytic triad of the natural hydrolases. In addition, the 17E8 active site features a protonated Lys side chain which we believe functions as an "oxyanion hole" equivalent to stabilize the development of oxyanion character in the hydrolytic transition state. In contrast, the natural hydrolases accommodate oxyanion stabilization with H-bonding interactions from backbone amide NH groups. Together the data suggest a hydrolytic mechanism similar to the natural enzymes involving nucleophilic attack by the active site Ser. We are currently testing this hypothesis with mechanistic probes, site-directed mutagenesis, and further structural characterization.