Essential histidyl residues of ferredoxin-NADP+ oxidoreductase revealed by diethyl pyrocarbonate inactivation.
Essential histidyl residues of ferredoxin-NADP+ oxidoreductase revealed by diethyl pyrocarbonate inactivation.
复制标题
通过焦碳酸二乙酯失活揭示铁氧还蛋白-NADP氧化还原酶的必需组氨酰残基。
作者：
N. Carrillo;R. Vallejos
二乙基碳酸盐酸盐抑制铁氧还蛋白NADP+氧化还原酶的dia氧化酶活性，其二阶速率常数为2 mm-1 x min-1在pH 7.0和20摄氏度下为2 mm-1 x min-1，表明由于甲氧氧基比衍生物的形成，在242 nm处的吸光度增加。羟胺可以恢复活性，灭活的pH曲线表明其PKA为6.8的残基参与。酪酶残基的衍生化也很明显，尽管对diaphorase活性没有影响。 NADP+和NADPH都保护了酶免于灭活，这表明修饰发生在核苷酸结合结构域或附近。在大约两个组氨酸残基被试剂阻断后，还原酶失去了所有diaphorase活性。在用NADP+作为保护剂的差异标记实验中，表明射氧化酶失活是由于每摩尔每摩尔酶仅阻断一个组酰基残基引起的。修饰的还原酶未结合吡啶核苷酸。在存在NADP+的情况下（即具有完全保存diaphorase活性）的黄素蛋白的修饰会导致细胞色素C还原酶活性的重大损害，二阶速率常数可使失活约0.5 mmm-1 x min-1。羟胺和光谱数据逆转表明，第二个残基也是组氨酸。铁氧还蛋白仅对这种抑制作用提供了轻微的保护。相反，酶的甲氧基不影响与亚硫硫蛋白的复合形成。用NADPH和还原的铁氧还蛋白对修饰的还原酶的氧化还原滴定表明，第二组氨酸可能位于FAD和FERREDOXIN之间的电子途径中。（抽象以250个单词截断）
Diethyl pyrocarbonate inhibited diaphorase activity of ferredoxin-NADP+ oxidoreductase with a second-order rate constant of 2 mM-1 X min-1 at pH 7.0 and 20 degrees C, showing a concomitant increase in absorbance at 242 nm due to formation of carbethoxyhistidyl derivatives. Activity could be restored by hydroxylamine, and the pH curve of inactivation indicated the involvement of a residue having a pKa of 6.8. Derivatization of tyrosyl residues was also evident, although with no effect on the diaphorase activity. Both NADP+ and NADPH protected the enzyme against inactivation, suggesting that the modification occurred at or near the nucleotide binding domain. The reductase lost all of its diaphorase activity after about two histidine residues had been blocked by the reagent. In differential-labeling experiments with NADP+ as protective agent, it was shown that diaphorase inactivation resulted from blocking of only one histidyl residue per mole of enzyme. Modified reductase did not bind pyridine nucleotides. Modification of the flavoprotein in the presence of NADP+, i.e., with full preservation of diaphorase activity, resulted in a significant impairment of cytochrome c reductase activity, with a second-order rate constant for inactivation of about 0.5 mM-1 X min-1. Reversal by hydroxylamine and spectroscopic data indicated that this second residue was also a histidine. Ferredoxin afforded only slight protection against this inhibition. Conversely, carbethoxylation of the enzyme did not affect complex formation with the ferrosulfoprotein. Redox titration of the modified reductase with NADPH and with reduced ferredoxin suggested that the second histidine might be located in the electron pathway between FAD and ferredoxin.(ABSTRACT TRUNCATED AT 250 WORDS)
铁氧还蛋白-NADP 氧化还原酶的结构和 NADP 结合位点的位置。
影响因子：
5.6
作者：
Sheriff,S;Herriott,JR
通讯作者：
Herriott,JR
DOI：
10.1073/pnas.78.11.6749
发表时间：
1981-11
影响因子：
11.1
作者：
S. Vik;Y. Hatefi
通讯作者：
S. Vik;Y. Hatefi
丁二酮、苯基乙二醛和焦碳酸二乙酯对 D(--)-β-羟基丁酸脱氢酶的抑制作用。
影响因子：
2.9
作者：
Phelps,DC;Hatefi,Y
通讯作者：
Hatefi,Y