pKa Coupling at the Intein Active Site: Implications for the Coordination Mechanism of Protein Splicing with a Conserved Aspartate

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• 作者：Zhenming Du ; Yuchuan Zheng ; Melissa Patterson ; Yangzhong Liu ; Chunyu Wang
• 刊名：Journal of the American Chemical Society
• 出版年：2011
• 出版时间：July 6, 2011
• 年：2011
• 卷：133
• 期：26
• 页码：10275-10282
• 全文大小：1014K
• 年卷期：v.133,no.26(July 6, 2011)
• ISSN：1520-5126

文摘

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鈥揝 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.