Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
Nucleic Acids Res. 1990 Oct 25;18(20):6083-8.

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

Author information

  • 1Katedra Biochemii, Akademia Rolnicza, PoznaƄ, Poland.

Abstract

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.

PMID:
2172926
[PubMed - indexed for MEDLINE]
PMCID:
PMC332409
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Icon for HighWire Icon for PubMed Central
    Loading ...
    Write to the Help Desk