N-15 NMR-SPECTROSCOPY OF HYDROGEN-BONDING INTERACTIONS IN THE ACTIVE-SITE OF SERINE PROTEASE - EVIDENCE FOR A MOVING HISTIDINE MECHANISM
N-15 NMR-SPECTROSCOPY OF HYDROGEN-BONDING INTERACTIONS IN THE ACTIVE-SITE OF SERINE PROTEASE - EVIDENCE FOR A MOVING HISTIDINE MECHANISM
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DOI：
10.1021/bi00371a070
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发表时间：
1986-11-18
期刊：
影响因子：
2.9
通讯作者：
BACHOVCHIN, WW
中科院分区：
文献类型：
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作者：
BACHOVCHIN, WW
氮-15 NMR光谱已被用于研究涉及α-环己基的催化三联体中的组氨酰基残基的氢键相互作用。在静息酶中和在与苯甲磺酰氟和氟磷酸二异丙酯形成的过渡态或四面体中间体类似物复合物中的裂解蛋白酶。15 N位移表明，强氢键连接的活性位点组氨酸和丝氨酸残基在溶液中的休息酶。这一结果与α-β的X射线衍射数据的解释不一致。裂解蛋白酶和其他丝氨酸蛋白酶的氨基酸序列，这表明丝氨酸和组氨酸残基相距太远，并且没有正确对齐以形成氢键。此外，氮-15的位移表明，在低pH值的组氨酸咪唑环的质子化的过渡态或四面体的中间体类似物与苯基甲磺酰氟和氟磷酸二异丙酯形成的复合物触发破坏的组氨酸-组氨酸氢键。这些结果表明，催化机制涉及直接运动的活性位点组氨酰残基的咪唑环。
Nitrogen-15 NMR spectroscopy has been used to study the hydrogen-bonding interactions involving the histidyl residue in the catalytic triad of .alpha.-lytic protease in the resting enzyme and in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate. The 15N shifts indicate that a strong hydrogen bond links the active site histidine and serine residues in the resting enzyme in solution. This result is at odds with interpretations of the X-ray diffraction data of .alpha.-lytic protease and of other serine proteases, which indicate that the serine and histidine residues are too far apart and not properly aligned for the formatin of a hydrogen bond. In addition, the nitrogen-15 shifts demonstrate that protonation of the histidine imidazole ring at low pH in the transition-state or tetrahedral intermediate analogue complexes formed with phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate triggers the disruption of the aspartate-histidine hydrogen bond. These results suggest a catalytic mechanism involving directed movement of the imidazole ring of the active site histidyl residue.