Retention of configuration in the action of human plasma 3'-exonuclease on oligo(deoxynucleoside phosphorothioate). A new method for assignment of absolute configuration at phosphorus in isotopomeric deoxyadenosine 5'-O-[(18)O]phosphorothioate.
A new method of analysis has allowed the exonucleolytic cleavage by human 3'-exonuclease to be determined. Hydrolysis by human plasma 3'-exonuclease proceeds with retention of configuration at phosphorus. The new method determines the sense of chirality at phosphorus in isotopomeric adenosine 5'-O-[(18)O]phosphorothioates. This is based on stereospecific two-step conversion of the mono-thionucleotide into the corresponding deoxyadenosine 5'-O-alpha-[(18)O]thiotriphosphate, followed by the use of terminal deoxyribonucleotidyl transferase and MALDI TOF mass spectrometry of the resulting elongated primer. Retention of configuration in the reaction of plasma 3'-exonuclease implies a two-step mechanism with two displacements on phosphorus. Inversion at each step leads to overall retention.
Koziołkiewicz
Maria
M
Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Sienkiewicza 112, 90-363 Łódź, Poland.
Owczarek
Alina
A
Wójcik
Marzena
M
Domański
Krzysztof
K
Guga
Piotr
P
Stec
Wojciech J
WJ
D003839:Deoxyadenosines / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D005092:Exonucleases / Q000097:blood / Q000737:chemistry*; D006801:Humans; D008968:Molecular Conformation; D010758:Phosphorus / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013873:Thionucleotides
D003839:Deoxyadenosines; D013873:Thionucleotides; C459740:deoxyadenosine 5'-O-((18)O)phosphorothioate; D010758:Phosphorus; D005092:Exonucleases; C022650:spleen exonuclease