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Mechanistic Studies of the Radical Transport Pathway in Aminotyrosine-Substituted CIass la Ribonucleotide Reductase
Başlık:
Mechanistic Studies of the Radical Transport Pathway in Aminotyrosine-Substituted CIass la Ribonucleotide Reductase
Yazar:
Lee, Wankyu, author.
Yazar Ek Girişi:
Genel Not:
Source: Dissertation Abstracts International, Volume: 79-10(E), Section: B.
Advisors: JoAnne Stubbe.
Özet:
Ribonucleotide reductase (RNR) catalyzes the reduction of nucleotides to 2'-deoxynucleotides. The focus of this thesis is the E. coli class Ia RNR, which is comprised of two homodimeric subunits, alpha2 and beta2, forming an active alpha2beta2 complex. The beta2 subunit harbors the stable diferric-tyrosyl radical cofactor (Y122)• that reversibly oxidizes the active site cysteine (C439) in alpha2. This oxidation requires a long-range radical transport (RT) pathway consisting of proton-coupled electron transfer (PCET) events through redox-active aromatic amino acid residues: Y122• ↔ [W48] ↔ Y356 in beta2 to Y731 ↔ Y730 ↔ C439 in alpha2. Once formed, the transient C439• initiates nucleotide reduction. Both the long-range oxidation and the nucleotide reduction chemistries are kinetically masked by rate-limiting protein conformational change(s). To overcome this conformational change, the unnatural amino acid probe 3-aminotyrosine (NH2Y) has been site-specifically incorporated at multiple positions (Y356, Y731, Y730) into the RT pathway.
Herein, the NH2Y probe is characterized as pertaining to the previously demonstrated ability for NH2Y-incorporated RNR (NH 2Y-RNR) to form product. The reduction potential of NH2Y produces a thermodynamic barrier that RNR cannot overcome. To explain NH 2Y-RNR activity, mass spectrometry was used for relative quantitation of contaminating wt-RNR in the NH2Y-RNR, lending credence to the fact that the NH2Y-RNRs are actually inactive. These results provide clarity to the long-standing mystery behind the low activities of the NH 2Y-RNRs.
The use of the NH2Y probe to generate stable radicals on the RT pathway has revealed further remarkable insight, demonstrating a hydrogen bonding network in the alpha2 subunit by employing advanced EPR methods on NH2Y730• and NH2Y731•. The evidence for a collinear PCET mechanism is provided with the NH2 Y730/Y731F and NH2Y731/C 439A mutants. Mutation of an R411 to alanine in alpha2 allowed the detection of a "flipped" NH2Y731 • conformation using advanced EPR techniques. Herein, photo cross-linked RNR is studied by tandem mass spectrometry (MS/MS). The study of a photo cross-linked alpha2 beta2 complex using a 4-N-maleimido-benzophenone covalently attached to the C-terminal tail of beta2 yielded no photo cross-linked peptides. These studies taken together provide additional insight at the alphabeta interface and provide additional tools to study this interaction. (Copies available exclusively from MIT Libraries, libraries.mit.edu/docs - docs mit.edu).
Notlar:
School code: 0753
Tüzel Kişi Ek Girişi:
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(687486.1) | 687486-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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