P alpha-chiral phosphorothioate analogues of bis(5'-adenosyl)tetraphosphate (Ap4A); their enzymatic synthesis and degradation.
Synthesis of Sp and Rp diastereomers of Ap4A alpha S has been characterized in two enzymatic systems, the lysyl-tRNA synthetase from Escherichia coli and the Ap4A alpha, beta-phosphorylase from Saccharomyces cerevisiae. The synthetase was able to use both (Sp)ATP alpha S and (Rp)ATP alpha S as acceptors of adenylate thus yielding corresponding monothioanalogues of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S. No dithiophosphate analogue was formed. Relative synthetase velocities of the formation of Ap4A,(Sp) Ap4A alpha S and (Rp)Ap4A alpha S were 1:0.38:0.15, and the computed Km values for (Sp)ATP alpha S and (Rp)ATP alpha S were 0.48 and 1.34 mM, respectively. The yeast Ap4A phosphorylase synthesized (Sp)Ap4A alpha S and (Rp)Ap4A alpha S using adenosine 5'-phosphosulfate (APS) as source of adenylate. The adenylate was accepted by corresponding thioanalogues of ATP. In that system, relative velocities of Ap4A, (Sp)Ap4A alpha S and (Rp)Ap4A alpha S formation were 1:0.15:0.60. The two isomeric phosphorothioate analogues of Ap4A were tested as substrates for the following specific Ap4A-degrading enzymes: (asymmetrical) Ap4A hydrolase (EC 3.6.1.17) from yellow lupin (Lupinus luteus) seeds hydrolyzed each of the analogues to AMP and the corresponding isomer of ATP alpha S; (symmetrical) Ap4A hydrolase (EC 3.6.1.41) from E. coli produced ADP and the corresponding diastereomer of ADP alpha S; and Ap4A phosphorylase (EC 2.7.7.53) from S. cerevisiae cleaved the Rp isomer only at the unmodified end yielding ADP and (Rp)ATP alpha S whereas the Sp isomer was degraded non-specifically yielding a mixture of ADP, (Sp)ADP alpha S, ATP and (Sp)ATP alpha S. For all the Ap4A-degrading enzymes, the Rp isomer of Ap4A alpha S appeared to be a better substrate than its Sp counterpart; stereoselectivity of the three enzymes for the Ap4A alpha S diastereomers is 51, 6 and 2.5, respectively. Basic kinetic parameters of the degradation reactions are presented and structural requirements of the Ap4A-metabolizing enzymes with respect to the potential substrates modified at the Ap4A-P alpha are discussed.
Lazewska
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Katedra Biochemii, Akademia Rolnicza, Poznań, Poland.
Guranowski
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A
D017766:Acid Anhydride Hydrolases; D015226:Dinucleoside Phosphates / Q000138:chemical synthesis*; D004926:Escherichia coli / Q000201:enzymology; D007202:Indicators and Reagents; D007700:Kinetics; D008250:Lysine-tRNA Ligase / Q000378:metabolism*; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013873:Thionucleotides
D015226:Dinucleoside Phosphates; D007202:Indicators and Reagents; D013873:Thionucleotides; C020733:diadenosine tetraphosphate; D009713:Nucleotidyltransferases; C044740:diadenosine 5,5'''-P(1),P(4)-tetraphosphate alpha,beta-phosphorylase; D010744:Phosphoric Monoester Hydrolases; D017766:Acid Anhydride Hydrolases; C020023:bis(5'-nucleosyl)tetraphosphatase (asymmetrical); D008250:Lysine-tRNA Ligase