New Probe for Transient Substrate Radicals in Enzymic Catalysis

近年来，越来越多的证据表明，许多酶都是通过自由基机制进行的。根据辅因子化学的模型研究，大多数反应都涉及氧化还原辅因子，其中自由基中间体是可信的。最近提出了涉及氨基酸侧链上的自由基的新型机制。虽然已经有许多令人信服的间接证据表明蛋白质衍生的自由基从底物中提取了H原子，但到目前为止还没有直接的证据。来自真菌Dactylium Dendroids的半乳糖氧化酶催化伯醇与O2氧化，产生醛和过氧化氢。基于最近的EPR、共振拉曼和X射线晶体结构信息，以及对基于机理的失活底物类似物的广泛的初步研究，提出了一种新的催化半乳糖氧化酶的机理，其特征是乙醇CH2中的酪氨酸自由基以速度决定H原子的提取。半乳糖氧化酶催化的反应机理将在方案研究中进行研究。由此产生的详细反应机理应该成为使用蛋白质为中心的自由基从底物中提取氢原子来激活底物的范例。这些基本信息将有助于理解更多与生物医学相关的酶，如核苷酸还原酶，由于严格的底物特异性，这些酶更难研究。

In recent years evidence has been accumulating that many enzymes proceed through radical mechanisms. Most reactions involve redox cofactors for which radical intermediates are plausible based on model studies of cofactor chemistry. New types of mechanisms have been proposed recently involving radicals on amino acid side chains. Although there has been much compelling indirect evidence consistent with H-atom abstraction from substrates by protein-derived radicals, to date there has been no direct evidence. Galactose oxidase from the fungus Dactylium dendroides catalyzes the oxidation of primary alcohols with O2, producing aldehydes and H2O2. Based on recent EPR, resonance Raman, and X-ray crystallographic structural information and on extensive preliminary studies with mechanism-based inactivating substrate analogs, a new mechanism for galactose oxidase catalysis is proposed featuring rate-determining H-atom abstraction by a tyrosine radical from the alcohol CH2. The reaction mechanism of galactose oxidase catalysis will be studied in the proposal research. The resulting detailed reaction mechanism should become a paradigm for substrate activation using protein-centered radicals for H- atom abstraction from substrates. Such fundamental information will be useful in understanding more biomedically relevant enzymes, such as ribonucleotide reductases, which are more difficult to study due to stringent substrate specificities.