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Nucleic Acids Research, 1990, Vol. 18, No. 20 6083-6088
© 1990

ENZYMOLOGY

P{alpha}-chiral phosphorothioate analogues of bis(5'-adenosyl)tetraphosphate (Ap4A) their enzymatic synthesis and degradation

D. Lazewska and A. Guranowski

Katedra Biochemii, Akademia Rolnicza, ul Wolynnska 35, 60–637 Poznan, Poland

Received June 7, 1990. Accepted July 19, 1990.

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},ß-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 [EC] ) 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 [EC] ) from E. coli produced ADP and the corresponding diastereomer of ADP{alpha}S; and Ap4A phosphorylase (EC 2.7.7.53 [EC] ) 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-speciflcally yielding a mixture of ADP, (Sp)ADP{alpha}S, ATP and (Sp)ATP{alpha}S. For all the Ap4A-degrading enyzmes, 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.


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