tailieunhanh - Báo cáo khoa học: Structural requirements for Caenorhabditis elegans DcpS substrates based on fluorescence and HPLC enzyme kinetic studies

The activity of theCaenorhabditis elegansscavenger decapping enzyme (DcpS) on its natural substrates and dinucleotide cap analogs, modified with regard to the nucleoside base or ribose moiety, has been examined. All tested dinucleotides were specifically cleaved between b- and c-phosphate groups in the triphosphate chain. | Structural requirements for Caenorhabditis elegans DcpS substrates based on fluorescence and HPLC enzyme kinetic studies Anna Wypijewska1 Elzbieta Bojarska1 Janusz Stepinski1 Marzena Jankowska-Anyszka2 Jacek Jemielity1 Richard E. Davis3 and Edward Darzynkiewicz1 1 Division of Biophysics Institute of ExperimentalPhysics Faculty of Physics University of Warsaw Poland 2 Department of Chemistry University of Warsaw Poland 3 Department of Biochemistry and Molecular Genetics University of Colorado Schoolof Medicine Aurora CO USA Keywords enzyme kinetics fluorescence spectroscopy mRNA cap analogs mRNA degradation scavenger decapping enzymes Correspondence E. Bojarska Division of Biophysics Institute of ExperimentalPhysics Faculty of Physics University of Warsaw 93 Zwirki Wigury Ave. 02-089 Warsaw Poland Fax 48 22 554 0771 Tel 48 22 554 0779 E-mail elab@ Received 25 February 2010 revised 8 May 2010 accepted 12 May 2010 doi The activity of the Caenorhabditis elegans scavenger decapping enzyme DcpS on its natural substrates and dinucleotide cap analogs modified with regard to the nucleoside base or ribose moiety has been examined. All tested dinucleotides were specifically cleaved between b- and y-phosphate groups in the triphosphate chain. The kinetic parameters of enzymatic hydrolysis Km Vmax were determined using fluorescence and HPLC methods as complementary approaches for the kinetic studies of C. elegans DcpS. From the kinetic data we determined which parts of the cap structure are crucial for DcpS binding and hydrolysis. We showed that m32 2 7GpppG and m32 2 7GpppA are cleaved with higher rates than their monomethylated counterparts. However the higher specificity of C. elegans DcpS for monomethylguanosine caps is illustrated by the lower Km values. Modifications of the first transcribed nucleotide did not affect the activity regardless of the type of purine base. Our findings suggest C. elegans DcpS flexibility in the

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