Kinetics of C. Elegans DcpS Cap Hydrolysis Studied by Fluorescence Spectroscopy

J. Wierzchowski Department of Biophysics, University of Warmia and Mazury, Olsztyn, Poland

M. Pietrzak Department of Biophysics, University of Warmia and Mazury, Olsztyn, Poland

J. Stepinski Department of Biophysics, University of Warsaw, Warsaw, Poland

J. Jemielity Department of Biophysics, University of Warsaw, Warsaw, Poland

M. Kalek Department of Biophysics, University of Warsaw, Warsaw, Poland

E. Bojarska Department of Biophysics, University of Warsaw, Warsaw, Poland

M. Jankowska-Anyszka Department of Chemistry, University of Warsaw, Warsaw, Poland

R. E. Davis Department of Biochemistry and Molecular Genetics, University of Colorado Health Science Center, Aurora, Colorado, USA

E. Darzynkiewicz Department of Biophysics, University of Warsaw, Warsaw, Poland

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Abstract

DcpS (scavenger decapping enzyme) from nematode C. elegans readily hydrolyzes both monomethyl- and trimethylguanosine cap analogues. The reaction was followed fluorimetrically. The marked increase of fluorescence intensity after the cleavage of pyrophosphate bond in dinucleotides was used to determine K_m and V_maxvalues. Kinetic parameters were similar for both classes of substrates and only slightly dependent on pH. The hydrolysis was strongly inhibited by methylene cap analogues (m⁷Gp(CH₂)ppG and m⁷Gpp(CH₂)pG) and less potently by ARCA (m^{7,3′ O}GpppG).

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Kinetics of C. Elegans DcpS Cap Hydrolysis Studied by Fluorescence Spectroscopy

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Kinetics of C. Elegans DcpS Cap Hydrolysis Studied by Fluorescence Spectroscopy

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