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Catalytic mechanism of alpha-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling.

Barabás, Orsolya and Németh, Veronika and Bodor, Andrea and Perczel, András and Rosta, Edina and Kele, Zoltán and Zagyva, Imre and Szabadka, Zoltán and Grolmusz, Vince I. and Vértessy G., Beáta (2013) Catalytic mechanism of alpha-phosphate attack in dUTPase is revealed by X-ray crystallographic snapshots of distinct intermediates, 31P-NMR spectroscopy and reaction path modelling. NUCLEIC ACIDS RESEARCH. pp. 1-14. ISSN 0305-1048

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Abstract

Enzymatic synthesis and hydrolysis of nucleoside phosphate compounds play a key role in various biological pathways, like signal transduction, DNA synthesis and metabolism. Although these processes have been studied extensively, numerous key issues regarding the chemical pathway and atomic movements remain open for many enzymatic reactions. Here, using the Mason-Pfizer monkey retrovirus dUTPase, we study the dUTPase-catalyzed hydrolysis of dUTP, an incorrect DNA building block, to elaborate the mechanistic details at high resolution. Combining mass spectrometry analysis of the dUTPase-catalyzed reaction carried out in and quantum mechanics/molecular mechanics (QM/MM) simulation, we show that the nucleophilic attack occurs at the alpha-phosphate site. Phosphorus-31 NMR spectroscopy (31P-NMR) analysis confirms the site of attack and shows the capability of dUTPase to cleave the dUTP analogue alpha,beta-imido-dUTP, containing the imido linkage usually regarded to be non-hydrolyzable. We present numerous X-ray crystal structures of distinct dUTPase and nucleoside phosphate complexes, which report on the progress of the chemical reaction along the reaction coordinate. The presently used combination of diverse structural methods reveals details of the nucleophilic attack and identifies a novel enzyme-product complex structure.

Item Type: Article
Subjects: Q Science / természettudomány > QH Natural history / természetrajz > QH301 Biology / biológia > QH3011 Biochemistry / biokémia
SWORD Depositor: MTMT SWORD
Depositing User: MTMT SWORD
Date Deposited: 04 Nov 2013 14:24
Last Modified: 04 Nov 2013 14:24
URI: http://real.mtak.hu/id/eprint/7140

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