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03월 10일 13시 이후 : 초록수정 불가능, 일정확인 및 검색만 가능
대한화학회 제105회 학술발표회 및 총회
An Emerging Protein Phosphatase - The Kinetic and Structural Study
2010년 2월 8일 19시 18분 58초
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금 11시 : 00분
단백질 과학의 생화학적 접근
건국대학교 응용생화학과, Korea
Phosphorylation and dephosphorylation of the C-terminal domain (CTD) of RNA polymerase II (Pol II) represent a critical regulatory checkpoint for transcription. Transcription initiation requires Fcp1/Scp1-mediated dephosphorylation of phospho-CTD. Fcp1 and Scp1 belong to a family of Mg2+ -dependent phosphoserine (P.Ser)/phosphothreonine (P.Thr)-specific phosphatases. We present the X-ray crystal structures of a dominant-negative form of human Scp1 (D96N mutant) bound to mono- and diphosphorylated peptides encompassing the CTD heptad repeat (Y1S2P3T4S5P6S7). Moreover, kinetic and thermodynamic analyses of Scp1-phospho-CTD peptide complexes support the structures determined. This combined structure-function analysis discloses the residues in Scp1 involved in CTD binding and its preferential dephosphorylation of P.Ser5 of the CTD heptad repeat. Moreover, these results provide a template for the design of specific inhibitors of Scp1 for the study of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg2+ ion and a DXDX(T/V) motif, both of which are signature features of HAD super family members. The first Asp of the motif is identified to be the nucleophile that is subjected for phosphorylation leading to a phosphoryl-aspartate intermediate. This high-energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. The proposed mechanism for Scp1 is widely accepted, however, the phosphoryl intermediate has never been captured directly in Scp1 or its closed family members. In the present study, we successfully captured the phosphoryl-aspartate intermediate in the crystal structure of Scp1 D206A mutant soaked with para-nitro phenyl phosphate (pNPP), providing confirmative evidence for the proposed two-step mechanism. Furthermore, steady-state kinetic analysis of a variety of Scp1 mutants revealed the Mg2+-coordinating function of Asp206, a conserved residue in HAD superfamily whose role was not noticed before. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play a structural and mechanistic role. This study laid the foundation for future studies on the mechanism of Scp1 and other HAD family members.
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