The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition
The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition
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DOI：
10.1073/pnas.0400973101
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发表时间：
2004-05-25
影响因子：
11.1
通讯作者：
Nishino, T
中科院分区：
文献类型：
--
作者：
Okamoto, K;Matsumoto, K;Nishino, T
钼在生物学中分布广泛，通常作为单核金属中心存在于许多催化氧原子转移的酶的活性位点中。钼羟化酶与催化羟基化反应的其他生物系统不同，因为掺入产物中的氧原子源自水而不是分子氧。在这里，我们展示了黄嘌呤氧化还原酶与慢速底物的羟基化反应中关键中间体的晶体结构，其中形成了产物的碳-氧键，但产物仍然与钼络合。该中间体在大约 640 nm 处显示出稳定的宽电荷转移带。配合物的晶体结构表明催化不稳定的Mo-OH氧已与底物的碳原子形成键。此外，氧化酶的Mo双键S基团已质子化，在钼中心还原时提供Mo-SH。与之前的分配相反，我们发现最后一个配体位于方锥体金属配位球的赤道位置，而不是顶端位置。目前的结构中不存在通常在酶活性位点中看到的水分子，这可能解释了该中间体对氢氧化物配体置换的稳定性。
Molybdenum is widely distributed in biology and is usually found as a mononuclear metal center in the active sites of many enzymes catalyzing oxygen atom transfer. The molybdenum hydroxylases are distinct from other biological systems catalyzing hydroxylation reactions in that the oxygen atom incorporated into the product is derived from water rather than molecular oxygen. Here, we present the crystal structure of the key intermediate in the hydroxylation reaction of xanthine oxidoreductase with a slow substrate, in which the carbon-oxygen bond of the product is formed, yet the product remains complexed to the molybdenum. This intermediate displays a stable broad charge-transfer band at approximate to640 nm. The crystal structure of the complex indicates that the catalytically labile Mo-OH oxygen has formed a bond with a carbon atom of the substrate. In addition, the Modouble bondS group of the oxidized enzyme has become protonated to afford Mo-SH on reduction of the molybdenum center. In contrast to previous assignments, we find this last ligand at an equatorial position in the square-pyramidal metal coordination sphere, not the apical position. A water molecule usually seen in the active site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide.