Functional properties of subunit interactions in human cytidine deaminase
Functional properties of subunit interactions in human cytidine deaminase
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DOI：
10.1093/protein/gzg117
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发表时间：
2003-12-01
期刊：
影响因子：
--
通讯作者：
Vita, A
中科院分区：
文献类型：
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作者：
Vincenzetti, S;De Sanctis, G;Vita, A
一个亚基间相互作用的活性位点的研究已进行野生型胞苷脱氨酶（CDA）和突变酶F137 W/W113 F。F137与参与亚基相互作用的枯草芽孢杆菌CDA F125同源。在SDS的存在下，野生型人CDA解离成无酶活性的单体，没有中间形式通过非合作转变。灭活单体的大量透析或稀释可完全恢复活性。圆二色性测量表明，每个亚基的二级/三级结构组织不受SDS浓度的影响，而突变Phe/Trp导致四级结构减弱。强的人CDA竞争性抑制剂5-fluorozebularine的存在不利于四聚体解离成野生型CDA中的亚基，但不是在突变酶F137 W/W113 F中。酪氨酸荧光的情况下，高得多的量子产率的双突变蛋白质光谱表明蛋白质亚基之间的能量转移效应的发生。对B的晶体学研究证实了这一假设。其中显示三个不同的亚基与四个活性位点中的每一个的形成同时发生，并且与人CDA F137同源的F125位于有助于活性位点形成的两个不同亚基之间的界面处。
An intersubunit interactions study related to the active site has been performed on the wild-type cytidine deaminase (CDA) and on the mutant enzyme F137W/ W113F. F137 is the homologous to the Bacillus subtilis CDA F125 involved in the subunit interactions. In the presence of SDS, wild-type human CDA dissociates into enzymatically inactive monomers without intermediate forms via a non-cooperative transition. Extensive dialysis or dilution of the inactivated monomers restores completely the activity. Circular dichroism measurements show that the secondary/tertiary structure organization of each subunit is unaffected by the SDS concentration, while the mutation Phe/Trp causes weakening in quaternary structure. The presence of the strong human CDA competitive inhibitor 5-fluorozebularine disfavours dissociation of the tetramer into subunits in the wild-type CDA, but not in mutant enzyme F137W/ W113F. The absence of tyrosine fluorescence and the much higher quantum yield of the double mutant protein spectrum suggest the occurrence of an energy transfer effect between the protein subunits. This assumption is confirmed by the crystallographic studies on B. subtilis in which it is shown that three different subunits concur with the formation of each of the four active sites and that F125, homologous to the human CDA F137, is located at the interface between two different subunits contributing to the formation of active site.