PROTON TRANSFER ESSENTIAL TO CATALYTIC ACTIVATION IN SOD

描述：（改编自申请人的摘要） 超氧化物歧化酶（Fe-SOD）催化超氧化物转化为 分子氧和过氧化氢，从而防止老化和退化 疾病 SOD的催化活性依赖于它提供质子的能力 Eo介于超氧离子的还原和氧化之间。 我们 提出核磁共振实验，以确定参与质子转移的残基， 通过静电相互作用和活性位点氢的氧化还原调节 键合网络 还原和氧化SOD中Tyr 34的pKs比较， 将揭示Tyr 34是否提供质子 在Fe氧化或结合时与底物结合。 如果pK不下降 在氧化时，将鉴定出配位溶剂而不是Tyr 34 作为质子供体。 的pK之间的差异， 活性位点可电离的氨基酸Tyr 34，His 30和Tyr 76在两个 氧化态将揭示质子化状态的程度， 这些都与铁的氧化态有关。 因此，我们将阐明 质子转移与底物结合和电子转移的耦合。 活性中心的氢键网络对催化剂的活性起着重要的作用。 活性部位的热力学和动力学能力。 的 拟议的工作将确定与电子有关的氢键中的质子 转移、底物结合和质子转移（并因此催化 活性）的功能H/D标记。 中心Gln残基的替换 连接到活性位点的氢键网络， 区分活性位点的结构扰动（在 用其氢键模拟中性His替代Gln），和 在随后的His质子化后氢键的破坏。 通过比较交换率，可以确定受影响的氢键。 残基69的氢键键合官能度的变化，因此 活性位点氢键网络。 因此，我们将阐明质子转移与电子转移的耦合， 探索氢键网络的本质和意义。 两者都是 普遍存在的，经常被提出但知之甚少的酶催化特征。 核磁共振直接观察质子的能力非常适合这个问题。

DESCRIPTION: (adapted from applicant's abstract) The Fe-containing superoxide dismutases (Fe-SODs) catalyze conversion of superoxide to dioxygen and hydroge peroxide, thus forestalling aging and degenerative diseases. SOD's catalytic activity rests on its ability to provide protons and Eo between those of reduction and oxidation of superoxide ion. We propose NMR experiments to identify residues involved in proton transfer and redox tuning via electrostatic interactions and the active site hydrogen bond network. Comparison of the pKs of Tyr 34 in reduced and oxidized SOD, with and without substrate analogs bound will reveal whether Tyr 34 donates a proton to substrate upon Fe oxidation or upon binding. If the pK does not drop upon oxidation then coordinated solvent instead of Tyr 34 will be identified as the proton donor in that step. The difference between the pKs of the active site ionizable amino acids Tyr 34, His 30 and Tyr 76 in the two oxidation states will reveal the extent to which the protonation state of any of these are coupled to Fe's oxidation state. Thus we will elucidate coupling of proton transfer to substrate binding and electron transfer. Hydrogen bonding networks in the active site exert an important effect on both the thermodynamic and kinetic capabilities of the active site. The proposed work will identify protons in hydrogen bonds related to electron transfer, substrate binding and proton transfer (and thus catalytic activity) by functional H/D labeling. Replacement of a Gln residue central to the active site hydrogen bond network with a His will allow us to distinguish between structural perturbation of the active site (upon replacement of Gln with it's hydrogen bonding mimic neutral His), and disruption of hydrogen bonding upon subsequent protonation of His. Comparison of the exchange rates will identify hydrogen bonds affected by a change in the hydrogen bonding functionality of residue 69, and thus the active site hydrogen bond network. Thus we will elucidate coupling of proton transfer to electron transfer and probe the nature and significance of hydrogen bond networks. Both are ubiquitous, oft-proposed but poorly understood features of enzyme catalysis. NMR's ability to directly observe protons suits it ideally to the problem.