PROTON TRANSFER ESSENTIAL TO CATALYTIC ACTIVATION IN SOD

描述：(改编自申请人的摘要)含铁 超氧化物歧化酶(Fe-SODS)催化超氧化物歧化为 氧气和过氧化氢，从而预防衰老和退化 疾病。超氧化物歧化酶的催化活性取决于其提供质子的能力 EO介于超氧阴离子还原和氧化之间。我们 建议进行核磁共振实验以确定参与质子转移的残基和 通过静电相互作用和活性位氢进行氧化还原调节 邦德网络。 酪氨酸34在还原和氧化两种状态下的蛋白水解酶活性比较 没有底物的类似物结合将揭示Tyr 34是否捐赠了质子 在铁氧化或结合时形成衬底。如果PK不掉落 氧化后，将确定配位溶剂而不是TYR 34 作为这一步中的质子供体。两种产品的PKS之间的差异 活性部位可电离氨基酸分别为Tyr 34、His 30和Tyr 76 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，我们将澄清 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反应有重要影响 活性中心的热力学和动力学能力。这个 拟议的工作将识别与电子有关的氢键中的质子 转移、底物结合和质子转移(从而催化 活性)，通过功能H/D标记。谷氨酰胺残基中心的替换 到活性部位的氢键网络与His的结合将允许我们 区分活性部位的结构扰动(在 用其氢键模拟中性His取代Gln)，以及 在随后的组氨酸质子化时氢键的破坏。 汇率的比较将确定氢键受 残基69的氢键官能度的变化，从而 活性中心氢键网络。 因此，我们将阐明质子转移到电子转移的耦合以及 探讨氢键网络的性质和意义。两者都是 无处不在的，经常被提出但鲜为人知的酶催化的特征。 核磁共振直接观测质子的能力非常适合解决这个问题。

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.