Synthesis of (Rp,Rp)-P1,P4-Bis(5'-adenosyl)-1[17O,18O2],4[17O,18O2] tetraphosphate from (Sp,Sp)-P1,P4-Bis(5'-adenosyl)-1[thio-18O2], 4[thio-18O2]tetraphosphate with retention at phosphorus and the stereochemical course of hydrolysis by the unsymmetrical Ap4A phosphodiesterase from lupin seeds.
The three stereoisomers of P1,P4-bis(5'-adenosyl)-1,4-dithiotetraphosphate have been synthesized and their 31P NMR spectra investigated. The effect of temperature on the circular dichroic spectrum of the (Sp,Sp)-stereoisomer shows that unstacking of the molecule occurs as the temperature is raised. Treatment of the (Sp,Sp)-stereoisomer with cyanogen bromide in [18O]water leads to substitution of sulfur by 18O with predominant retention of configuration at P1 and P4. (Sp,Sp)-P1,P4-Bis(5'-adenosyl)-1[thio-18O2],4[thio-18O2]tetraphosphate was synthesized and on treatment with cyanogen bromide in [17O]water gave (Rp,Rp)-P1,P4-bis(5'-adenosyl)-1[17O,18O2],4[17O,18O2]tetraphosphate. Hydrolysis by unsymmetrical Ap4A phosphodiesterase from lupin seeds gave (Rp)-5'-[16O,17O,18O]AMP. The reaction therefore proceeds with inversion of configuration at phosphorus, indicating that the enzyme-catalyzed displacement by water occurs by a direct "in-line" mechanism.
Dixon
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RM
Dyson Perrins Laboratory, Oxford University, Oxford, United Kingdom.
Lowe
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D017766:Acid Anhydride Hydrolases; D015226:Dinucleoside Phosphates / Q000138:chemical synthesis* / Q000378:metabolism; D007887:Fabaceae / Q000201:enzymology; D006868:Hydrolysis; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods; D009682:Magnetic Resonance Spectroscopy; D010103:Oxygen Isotopes; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010946:Plants, Medicinal; D012639:Seeds / Q000201:enzymology*; D013237:Stereoisomerism; D013873:Thionucleotides / Q000138:chemical synthesis* / Q000378:metabolism
D015226:Dinucleoside Phosphates; D007202:Indicators and Reagents; D010103:Oxygen Isotopes; D013873:Thionucleotides; C058477:P(1),P(4)-bis(5'-adenosyl)-1,4-dithiotetraphosphate; D010744:Phosphoric Monoester Hydrolases; D017766:Acid Anhydride Hydrolases; C020023:bis(5'-nucleosyl)tetraphosphatase (asymmetrical)