Oxygen pressurized X-ray crystallography:: Probing the dioxygen binding site in cofactorless urate oxidase and implications for its catalytic mechanism
Oxygen pressurized X-ray crystallography:: Probing the dioxygen binding site in cofactorless urate oxidase and implications for its catalytic mechanism
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DOI：
10.1529/biophysj.107.122184
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发表时间：
2008-09-01
影响因子：
3.4
通讯作者：
Prange, Thierry
中科院分区：
文献类型：
--
作者：
Colloc'h, Nathalie;Gabison, Laure;Prange, Thierry
氧结合蛋白中分子氧位点的定位是一项重要的实验任务，通常通过间接方法进行，例如使用氙或卤阴离子作为氧探针。在这项研究中，一个简单的方法，在高压纯氧下的X射线晶体学的基础上已经开发出来。尿酸氧化酶（UOX）是一种无辅因子的酶，在分子氧存在下催化尿酸氧化为5-羟基异尿酸。UOX晶体与其天然基质的竞争性抑制剂复合，经受1.0、2.5或4.0 MPa的气态氧的增加压力。结果清楚地表明，分子氧结合在活性位点内的水分子通常被观察到的位置，但不结合在已经表征的氙的特定疏水口袋。此外，在大量过量的氯化物（NaCl）存在下结晶UOX表明，一个氯离子与氧处于相同的位置。分子氧亲水性环境（天冬酰胺，组氨酸和苏氨酸残基），其在氙结合位点内的缺失，以及其与水分子或氯离子相同的位置表明分子氧位点主要是极性的。分子氧的位置上的机制的含义进行了讨论，相对于实验建议的瞬态中间体在反应级联。
The localization of dioxygen sites in oxygen-binding proteins is a nontrivial experimental task and is often suggested through indirect methods such as using xenon or halide anions as oxygen probes. In this study, a straightforward method based on x-ray crystallography under high pressure of pure oxygen has been developed. An application is given on urate oxidase (UOX), a cofactorless enzyme that catalyzes the oxidation of uric acid to 5-hydroxyisourate in the presence of dioxygen. UOX crystals in complex with a competitive inhibitor of its natural substrate are submitted to an increasing pressure of 1.0, 2.5, or 4.0 MPa of gaseous oxygen. The results clearly show that dioxygen binds within the active site at a location where a water molecule is usually observed but does not bind in the already characterized specific hydrophobic pocket of xenon. Moreover, crystallizing UOX in the presence of a large excess of chloride (NaCl) shows that one chloride ion goes at the same location as the oxygen. The dioxygen hydrophilic environment (an asparagine, a histidine, and a threonine residues), its absence within the xenon binding site, and its location identical to a water molecule or a chloride ion suggest that the dioxygen site is mainly polar. The implication of the dioxygen location on the mechanism is discussed with respect to the experimentally suggested transient intermediates during the reaction cascade.