Biology Department, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.
J Am Chem Soc. 2011 Jul 6;133(26):10275-82. doi: 10.1021/ja203209f. Epub 2011 Jun 9.
Protein splicing is a robust multistep posttranslational process catalyzed by inteins. In the Mtu RecA intein, a conserved block-F aspartate (D422) coordinates different steps in protein splicing, but the precise mechanism is unclear. Solution NMR shows that D422 has a strikingly high pK(a) of 6.1, two units above the normal pK(a) of aspartate. The elevated pK(a) of D422 is coupled to the depressed pK(a) of another active-site residue, the block-A cysteine (C1). A C1A mutation lowers the D422 pK(a) to normal, while a D422G mutation increases the C1 pK(a) from 7.5 to 8.5. The pK(a) coupling and NMR structure determination demonstrate that protonated D422 serves as a hydrogen bond donor to stabilize the C1 thiolate and promote the N-S acyl shift, the first step of protein splicing. Additionally, in vivo splicing assays with mutations of D422 to Glu, Cys, and Ser show that the deprotonated aspartate is essential for splicing, most likely by deprotonating and activating the downstream nucleophile in transesterification, the second step of protein splicing. We propose that the sequential protonation and deprotonation of the D422 side chain is the coordination mechanism for the first two steps of protein splicing.
蛋白质剪接是由内肽酶催化的一种强大的多步翻译后过程。在 Mtu RecA 内肽酶中，一个保守的块-F 天冬氨酸 (D422) 协调蛋白质剪接的不同步骤，但确切的机制尚不清楚。溶液 NMR 表明，D422 的 pK(a) 值高达 6.1，比天冬氨酸的正常 pK(a) 值高两个单位。D422 的高 pK(a) 值与另一个活性位点残基块-A 半胱氨酸 (C1) 的低 pK(a) 值相关联。C1A 突变将 D422 的 pK(a) 值降低到正常水平，而 D422G 突变将 C1 的 pK(a) 值从 7.5 增加到 8.5。pK(a) 值偶联和 NMR 结构测定表明，质子化的 D422 充当氢键供体，稳定 C1 硫醇盐并促进 N-S 酰基转移，这是蛋白质剪接的第一步。此外，对 D422 突变为 Glu、Cys 和 Ser 的体内剪接实验表明，去质子化的天冬氨酸对于剪接是必不可少的，最有可能通过去质子化并激活反式酯交换中的下游亲核试剂，这是蛋白质剪接的第二步。我们提出，D422 侧链的顺序质子化和去质子化是蛋白质剪接前两步的协调机制。
