【摘要】 本文通过测定丙酮酸氧化酶内源荧光谱和荧光偏振的变化研究了该酶在1—2200bar压力下的解离。研究结果表明,在压力作用下酶的辅基FAD不可逆地从酶分子上解离下来,并因此引起酶的失活;酶亚基在压力下的解离是可逆的,在5℃,pH7.6条件下,该酶的解离自由能⊿G°为29.89k cal/mol,解离标准体积变化⊿V°为-220ml/mol。脱辅基丙酮酸氧化酶的解离自由能为24.93k cal/mol,证明FAD对酶有稳定作用;⊿V°则为-153ml/mol,减少了近30％,表明FAD对亚基间的空间大小有很大贡献。经胰凝乳蛋白酶部分酶解所活化的酶的解离⊿G°和⊿V°均有所增加,底物丙酮酸亦有相同的影响。研究还表明,碱性pH条件能促进丙酮酸氧化酶的解离。在此研究中,我们也观察到了Weber和Ruan在乳酸脱氢酶、甘油醛-3-磷酸脱氢酶等研究中报道的"conformational drift"现象。更多还原

【Abstract】 The dissociation of pyruvate oxidase undtr hydrostatic pressure 1-2200 bar followed by measurement of intrinsic fluorescence spectra or polarization was studied in this paper. It was observed that the prosthetic group FAD of pyruvate oxidase molecule dissociated irreversibly under pressure; resulting in the inactivation of enzyme.But the pressure induced dissociation of the enzyme subunits is reversible. At 5℃ and pH 7.6, the free energy of dissociation for pyruvate oxidase, △G°, is 29.89 k cal/mol and the standard volume change upon dissociation △V°is -220 ml/mol. For apo-pyruvate oxidase, the free energy of dissociation △G° decreased to 24.93 k cal/mol and the standard vo- lume change upon dissociation △V° decreased to - 153 ml/mol comparing with the whole enzyme, that indicated FAD stabilizes the enzyme and makes a great contribution to the spatial volume between the subunits. After activation by limited proteolysis, both △G° and △V° of the enzyme increased. And the su-tstrate pyruvic acid gave a very similar effect. But the study of pH effect showed that alkaline pH promote the enzyme to dissociate. In this study we also observed "conformational drift’’phenomenon reported by Weber et al. in the studies of lactate dehydrogenase and D-glyceraldehyde-3-phosphate dehydrogenase etc.更多还原