Structural requirements for C. elegans DcpS substrates based on fluorescence and HPLC enzyme kinetic studies

• Published in: The FEBS journal Vol. 277; no. 14; pp. 3003 - 3013
• Main Authors: Wypijewska, Anna, Bojarska, Elzbieta, Stepinski, Janusz, Jankowska-Anyszka, Marzena, Jemielity, Jacek, Davis, Richard E., Darzynkiewicz, Edward
• Format: Journal Article
• Language: English
• Published: 08.06.2010
• DOI: 10.1111/j.1742-4658.2010.07709.x
• ISSN: 1742-464X

The activity of C. elegans scavenger decapping enzyme (DcpS) on its natural substrates and dinucleotide cap analogues modified in the nucleoside’s base or ribose moiety, has been examined. All tested dinucleotides were specifically cleaved between β and γ phosphate groups in the triphosphate chain. The kinetic parameters of enzymatic hydrolysis ( K m , V max ) were determined using fluorescence and HPLC methods, as complementary approaches for the kinetic studies of C. elegans DcpS. From the kinetic data, we have determined which parts of the cap structure are crucial for DcpS binding and hydrolysis. We show that m 3 2,2,7 GpppG and m 3 2,2,7 GpppA are cleaved with higher rates than their monomethylated counterparts. However, C. elegans DcpS higher specificity for MMG caps is illustrated by lower K m values. Modifications of the first transcribed nucleotide did not affect the activity, regardless of the type of purine base. Our finding suggests C. elegans DcpS flexibility in the first transcribed nucleoside-binding pocket. Moreover, while C. elegans DcpS accomodates bulkier groups in the N7 position (ethyl or benzyl) of the cap, either 2′- O -or 3′- O -methylations of the 7-methylguanosine result in two orders of magnitude reduction in hydrolysis.