Preparation of a mixture of nucleoside triphosphates suitable for use in synthesis of nucleotide phosphate sugars from ribonucleic acid using nuclease P1, a mixture of nucleoside monophosphokinases and acetate kinase.
This paper (1) describes the enzymatic synthesis of a mixture of adenosine, guanosine, cytidine, and uridine triphosphates (ATP, GTP, CTP, and UTP) from ribonucleic acid (RNA). RNA was hydrolyzed by nuclease P1 to a mixture of 5'-nucleoside monophosphates. This mixture was converted to the nucleoside triphosphates using a mixture of nucleoside monophosphate kinases and acetate kinase, with acetyl phosphate as the ultimate phosphoryl donor. The nucleoside monophosphokinases were extracted from brewer's yeast in a four-step procedure. The specific activity of the yeast enzyme preparation after gel permeation chromatography was sufficiently high that the yeast kinases could be immobilized in volumes that were practical for laboratory scale syntheses. Conversions from NMP to NTP in a mixture containing 0.34 mol of total nucleoside phosphates were: ATP, 90%; GTP, 90%; CTP, 60%; and UTP, 40%.
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
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Haynie
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Whitesides
D000084:Acetate Kinase / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D003570:Cytidine Triphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*; D014544:Uridine Triphosphate / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology
D012313:RNA; D003570:Cytidine Triphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000084:Acetate Kinase; D009703:Nucleoside-Phosphate Kinase; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014544:Uridine Triphosphate