Structural requirements for Caenorhabditis elegans DcpS substrates based on fluorescence and HPLC enzyme kinetic studies.
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 beta- and gamma-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 determined which parts of the cap structure are crucial for DcpS binding and hydrolysis. We showed that m(3)(2,2,7)GpppG and m(3)(2,2,7)GpppA 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 K(m) 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 first transcribed nucleoside-binding pocket. Moreover, although C. elegans DcpS accommodates bulkier groups in the N7 position (ethyl or benzyl) of the cap, both 2'-O- and 3'-O-methylations of 7-methylguanosine result in a reduction in hydrolysis by two orders of magnitude.
Wypijewska
Anna
A
Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Poland.
Bojarska
Elzbieta
E
Stepinski
Janusz
J
Jankowska-Anyszka
Marzena
M
Jemielity
Jacek
J
Davis
Richard E
RE
Darzynkiewicz
Edward
E
D000818:Animals; D055162:Biocatalysis; D017173:Caenorhabditis elegans / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D015226:Dinucleoside Phosphates / Q000378:metabolism; D006151:Guanosine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D011755:Pyrophosphatases / Q000737:chemistry / Q000378:metabolism*; D012314:RNA Cap Analogs / Q000378:metabolism*; D012315:RNA Caps / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
D029742:Caenorhabditis elegans Proteins; D015226:Dinucleoside Phosphates; D012314:RNA Cap Analogs; D012315:RNA Caps; D011994:Recombinant Proteins; D006151:Guanosine; C016578:7-methylguanosine; C017778:N(2),N(2),7-trimethylguanosine; C015139:diguanosine triphosphate; D011755:Pyrophosphatases; C509343:dcs-1 protein, C elegans