Zak O, Tam B, MacGillivray R T, Aisen P
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biochemistry. 1997 Sep 9;36(36):11036-43. doi: 10.1021/bi970628v.
Release of iron from transferrin, the iron-transporting protein of the circulation, is a concerted process involving remote amino acid residues as well as those at the two specific iron-binding sites of the protein. Previous studies of fluoresceinated transferrin have suggested Lys 569 as a kinetically active site in the C-terminal lobe of the protein. We have therefore turned to site-directed mutagenesis to investigate the role of Lys 569 in the release process at pH 5.6, the pH of the endosome where iron is transferred from transferrin to the iron-dependent cell. Mutation of positively charged Lys 569 to an uncharged Gln results in a protein in which release of iron from the mutated lobe to pyrophosphate is slowed by a factor of 15-20 and in which release kinetics switch from a complex saturation-linear to a simple saturation function. Acceleration of release by chloride is also substantially less than in native transferrin. When Lys 569 is replaced by a positively charged Arg, in contrast, observed release rates and chloride dependence are close to those of the native protein. The mechanism of release from the C-lobe site therefore appears to be sensitive to positive charge at position 569. Binding of chloride or other simple anion accelerates and is essential for release from the C-lobe; a muted response of K569Q to chloride concentration suggests that Lys 569 may function as a kinetically active anion-binding residue in the C-lobe. Despite the kinetic effects of the K569 mutation on iron release, rates of iron uptake by K562 cells from the C-lobes of native, K569Q, and K569R proteins are almost identical. In contrast to the C-lobe, iron release from the N-lobe is insensitive to charge at residue 233, the site in that lobe homologous to residue 569, with chloride retarding rather than accelerating release. K233, therefore, is not a kinetically active anion-binding site in the N-lobe. Release mechanisms differ substantially in the two lobes of transferrin despite the identity of ligands and their nearly identical arrangements in the lobes.
铁从转铁蛋白（循环中的铁转运蛋白）的释放是一个协同过程，涉及远距离氨基酸残基以及该蛋白两个特定铁结合位点处的氨基酸残基。先前对荧光素化转铁蛋白的研究表明，赖氨酸569是该蛋白C端叶中的一个动力学活性位点。因此，我们采用定点诱变来研究赖氨酸569在pH 5.6（即铁从转铁蛋白转移至铁依赖性细胞的内体的pH值）时的释放过程中的作用。带正电荷的赖氨酸569突变为不带电荷的谷氨酰胺后，会产生一种蛋白质，其中铁从突变叶释放至焦磷酸的速度减慢了15至20倍，且释放动力学从复杂的饱和-线性转变为简单的饱和函数。氯化物对释放的加速作用也远低于天然转铁蛋白。相反，当赖氨酸569被带正电荷的精氨酸取代时，观察到的释放速率和对氯化物的依赖性与天然蛋白接近。因此，从C叶位点的释放机制似乎对569位的正电荷敏感。氯化物或其他简单阴离子的结合会加速从C叶的释放，且对其释放至关重要；K569Q对氯化物浓度的反应减弱表明，赖氨酸569可能作为C叶中一个动力学活性的阴离子结合残基发挥作用。尽管K569突变对铁释放有动力学影响，但K562细胞从天然、K569Q和K569R蛋白的C叶摄取铁的速率几乎相同。与C叶不同，铁从N叶的释放对233位残基（该叶中与569位残基同源的位点）的电荷不敏感，氯化物会抑制而非加速释放。因此，K233不是N叶中一个动力学活性的阴离子结合位点。尽管两个叶中的配体相同且其排列几乎相同，但转铁蛋白两个叶中的释放机制却有很大差异。
