Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain.
Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain.
复制标题
通过吡啶核苷酸结合域中关键残基 (I184Y) 的定点诱变改变硫辛酰胺脱氢酶的特性。
DOI：
10.1021/bi00115a008
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
WilliamsJr,CH
中科院分区：
文献类型：
--
作者：
Maeda-Yorita,K;Russell,GC;Guest,JR;Massey,V;WilliamsJr,CH
1991年9月17日摘要：吡啶核苷酸与人红细胞谷胱甘肽还原酶（一种已知三维结构的酶）的结合需要Tyr197侧链的一些运动。此外，该侧链非常靠近FAD辅因子的异咯嗪环。同源酶大肠杆菌硫辛酰胺脱氢酶中的类似残基Iie184已通过定点突变改变为酪氨酸残基（I184Y）[Russell，GC，Allison，N.，威廉姆斯，C. H、小的，& Guest，J. R.（1989）Ann.NY Acad. sci. 573，429 - 431]。改变的酶的表征表明，吡啶核苷酸半反应的速率已显着降低，并已改变光谱特性，以模仿谷胱甘肽还原酶。因此，He 184被证明是在调节硫辛酰胺脱氢酶中的黄素的性质中的重要残基。在I184Y硫辛酰胺脱氢酶中，二氢硫辛酰胺/NAD+反应的周转率降低10倍，而在NADH/硫辛酰胺反应中降低2倍。氧化态的I184 Y在可见吸收光谱和圆二色性光谱的精细结构中显示出显著的变化，并且还显示出几乎完全猝灭的FAD荧光。因此，改变的酶的光谱特性类似于谷胱甘肽还原酶的光谱特性，而与野生型硫辛酰胺脱氢酶的光谱特性非常不同。另一方面，光谱证据并没有揭示在EH2水平的电荷转移稳定量的任何变化。停流数据表明，在还原I184 Y的NADH，第一步，还原的黄素，只有轻微放缓，但thesublesstwo-electrontransfer到二硫化物显着抑制。这允许直接观察假定的还原黄素种类。二氢硫辛酰胺还原I184 Y至EH2阶段未发生可检测到的改变，正如预期的那样，考虑到改变的位点。
Revised Manuscript Received September 17, 1991 abstract: The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197. Moreover, this side chain lies very close to theisoalloxazine ring of the FAD cofactor. The analogous residue, lie184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mu-tagenesis to a tyrosine residue (I184Y)[Russell, GC, Allison, N., Williams, C. H., Jr., & Guest, J. R.(1989) Ann. NY Acad. Sci. 573, 429-431]. Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reducedand that the spectral properties have been changedto mimic those of glutathione reductase. Therefore, He184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase. Turnover in the dihydrolipo-amide/NAD+ reaction is decreasedby 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase. The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching ofFAD fluorescence. The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-typelipoamide dehydrogenase. On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level. Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but thesubsequent two-electrontransfer to the disulfide is markedly inhibited. This allows direct observation of the presumed reduced flavin species. Reduction ofI184Y by dihydrolipoamide to the EH2 stage is not detectably altered, as expected given the site of the alteration.