MECHANISTIC ANALYSIS OF PROTON TRANSFER

通过从一个异构体上提取Calpha质子和在碳离子中间体或过渡态的相反面上质子化，双胺基异构体表异构体酶催化L，L-和D，L-中位二氨基异戊二酸酯的相互转化。虽然PLP非依赖的氨基酸外消旋酶家族的其他成员的结构尚未确定，但流感嗜血杆菌的dapF编码的酶已被克隆、表达、纯化和结晶，其三维结构被解析为2.7A。这274个氨基酸的酶具有一个新的折叠，两个催化半胱氨酸残基Cys73和Cys217位于分隔两个结构同源结构域的裂隙中。对单个突变体C73S和C217S的动力学和同位素研究将进一步表征半胱氨酸作为一般酸和碱的不同寻常的作用(而不是它在其他酶催化的反应中更典型的亲核作用)。这些实验还将分别指定在L、L和D、L和D、L和L、L方向上哪个半胱氨酸残基负责质子提取。此外，使用特定同位素标记和13C核磁共振测量两个半胱氨酸残基Cys73和Cys217的微观pk值，将揭示活性中心环境的细节，并将澄清稳定阶段pH研究的最新结果。此外，虽然没有可用的酶-底物或酶-抑制剂结构，但底物结合部位也将使用氢/氚酰胺交换和质谱仪进行分析。这些实验将进一步了解二氨基异构酶的作用机制，这种酶在辅因子非依赖的消旋酶中是不寻常的，具有独特的性质，包括平衡常数等于2。

Diaminopimelete epimerase catalyzes the interconversion of L,L-and D,L-meso-diaminopimelate, via Calpha proton abstraction from one isomer and protonation at the opposite face of the carbanionic intermediate or transition state. While no structures of other members of the PLP-independent amino acid racemase family have yet been determined, the dapF encoded enzyme from Haemophilus influenzae has been cloned, expressed, purified and crystallized, and the three- dimensional structure was solved to 2.7 A. This 274 amino acid enzyme exhibits a novel fold, and the two catalytic cysteine residues, Cys73 and Cys217, are located in a cleft separating the two structurally homologous domains. The proposed kinetic and isotopic studies of the single mutants, C73S and C217S, will further characterize the unusual role of the cysteine as a general acid and base (versus its more typical role as a nucleophile in other enzyme catalyzed reactions). These experiments will also assign which cysteine residue is responsible for proton abstraction in the L, L- >D,L and D,L->L,L directions, respectively. In addition, the measurement of the microscopic pK values of the two cysteine residues, Cys73 and Cys217, using specific isotopic labeling and 13C nuclear magnetic resonance, will reveal details of the active site environment and will clarify the recent results from the steady-stage pH studies. Further, while no enzyme-substrate or enzyme-inhibitor structure is available, the substrate-binding site will also be analyzed using hydrogen/deuterium amide exchange and mass spectrometry. These proposed experiments will further understanding of the mechanism of diaminopimelate epimerase which is unusual among the cofactor independent racemases with unique properties including an equilibrium constant equal to 2.