Yu B Z, Rogers J, Nicol G R, Theopold K H, Seshadri K, Vishweshwara S, Jain M K
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Biochemistry. 1998 Sep 8;37(36):12576-87. doi: 10.1021/bi9728607.
Calcium is required for the substrate binding and for the chemical step of the interfacial catalytic turnover cycle of pancreatic phospholipase A2 (PLA2), but not for the binding of the enzyme to the interface. The role of calcium and other divalent cations (C) is analyzed for the effect on the substrate binding and kcat* for the chemical step. The cofactor role of 3d-cations(II) (C) for the hydrolysis of dimyristoylphosphatidylmethanol (DMPM) vesicles is characterized as an equilibrium dissociation constant for the interfacial binary (EC) and ternary (ECL) complexes of PLA2 and substrate mimics (L). Of the cations(II) that promote the binding of a mimic to the enzyme at the interface (E*), only a subgroup supports the chemical step. For example, Cd, Zn, and Cu form ternary ECL complexes with kcat of <1 s-1, compared to the rate of >100 s-1 with Ca, Fe, Mn, Co, and Ni. Oxygen exchange from H218O to the products of hydrolysis of DMPM incorporates one 18O in myristate. Incorporation of the first and second 18O occurs during the incubation of both the products of hydrolysis in H218O with PLA2 and Ca, but not with Zn. The cation-dependent changes in the UV difference spectrum, associated with the formation of EC and ECL, suggest that the changes are mainly due to catalytic His-48, and possibly Tyr-52 and Tyr-73, and are different with Ca as opposed to Zn. These results and simulations suggest considerable plasticity in the calcium binding and catalytic site environment. It is proposed that the higher ground state stability of the ECS complex with the inhibitory cations increases the effective activation energy. For the chemical step, calcium coordinated with a nucleophilic water and the ester carbonyl oxygen facilitates the near-attack geometry in the ECaS, and the His-48.Asp-99 pair acts as a proton acceptor. As a prelude to establishing the catalytic mechanism, factors controlling the energetically demanding transition state are also discussed.
钙对于胰腺磷脂酶A2（PLA2）界面催化周转循环的底物结合和化学步骤是必需的，但对于酶与界面的结合则不是必需的。分析了钙和其他二价阳离子（C）对底物结合和化学步骤的催化常数（kcat*）的影响。3d阳离子（II）（C）对二肉豆蔻酰磷脂酰甲醇（DMPM）囊泡水解的辅助因子作用被表征为PLA2与底物模拟物（L）的界面二元（EC）和三元（ECL）复合物的平衡解离常数。在促进模拟物与界面处的酶（E*）结合的阳离子（II）中，只有一个亚组支持化学步骤。例如，镉、锌和铜形成三元ECL复合物，其kcat<1 s-1，而钙、铁、锰、钴和镍的速率>100 s-1。从H218O到DMPM水解产物的氧交换在肉豆蔻酸中掺入了一个18O。在水解产物与PLA2和钙一起孵育期间会发生第一个和第二个18O的掺入，但与锌一起孵育时则不会。与EC和ECL形成相关的紫外差光谱中阳离子依赖性变化表明，这些变化主要归因于催化性的组氨酸-48，可能还有酪氨酸-52和酪氨酸-73，并且钙与锌的情况不同。这些结果和模拟表明钙结合和催化位点环境具有相当大的可塑性。有人提出，ECS复合物与抑制性阳离子的较高基态稳定性增加了有效活化能。对于化学步骤，与亲核水和酯羰基氧配位的钙促进了ECaS中的近攻击几何结构，组氨酸-48-天冬氨酸-99对充当质子受体。作为建立催化机制的前奏，还讨论了控制能量需求高的过渡态的因素。
