Steinlein L M, Ligman C M, Kessler S, Ikeda R A
Department of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400, USA.
Biochemistry. 1998 Sep 29;37(39):13696-703. doi: 10.1021/bi980318s.
Human serum transferrin consists of two iron-binding lobes connected by a short peptide linker. While the high homology and structural similarity between the two halves of the molecule would suggest similar characteristics, it has been shown that the pH-dependent rate of release of iron from the N-terminal lobe is quite different from that of its C-terminal counterpart. This suggests that the N-lobe of human serum transferrin has a specific, pH-dependent, molecular mechanism for releasing iron. Sacchettini and co-workers using structural information have hypothesized that two lysines in the N-terminal lobe of ovotransferrin create a dilysine interaction and suggest that this is the trigger for pH-dependent iron release. To investigate this hypothesis, we used a Pichia pastoris expression system to produce large amounts of wild-type nTf, the single point mutants, nTfK206A (Lys 206 to alanine) and nTfK296A (Lys 296 to alanine), and the double mutant, nTfK206/296A. The purified recombinant proteins were then used to measure rates of iron release to pyrophosphate. It was found that the rate of iron release from all three mutant proteins at pH 5.7 (the pH at which nTf would normally release iron) was too slow to measure. Only when the pH was reduced to 5.0 could the rates of iron release from the mutant proteins be reliably determined. Although this precludes a direct comparison to wild-type nTf (the rate of iron release from nTf at pH 5.0 is too fast to measure), it implicates lysines 206 and 296 in the pH-dependent release of iron from nTf.
人血清转铁蛋白由两个通过短肽连接子相连的铁结合结构域组成。虽然分子两半部分之间的高度同源性和结构相似性表明具有相似的特性，但已表明铁从N端结构域释放的pH依赖性速率与其C端对应物有很大不同。这表明人血清转铁蛋白的N端结构域具有特定的、pH依赖性的铁释放分子机制。萨凯蒂尼及其同事利用结构信息推测，卵转铁蛋白N端结构域中的两个赖氨酸形成双赖氨酸相互作用，并表明这是pH依赖性铁释放的触发因素。为了研究这一假设，我们使用毕赤酵母表达系统大量生产野生型nTf、单点突变体nTfK206A（赖氨酸206突变为丙氨酸）和nTfK296A（赖氨酸296突变为丙氨酸）以及双突变体nTfK206/296A。然后使用纯化的重组蛋白测量铁释放到焦磷酸的速率。发现在pH 5.7（nTf正常释放铁的pH值）时，所有三种突变蛋白的铁释放速率都太慢而无法测量。只有当pH降至5.0时，才能可靠地测定突变蛋白的铁释放速率。虽然这排除了与野生型nTf的直接比较（nTf在pH 5.0时的铁释放速率太快而无法测量），但这表明赖氨酸206和296参与了nTf的pH依赖性铁释放。
