双（5'-腺苷）四磷酸（Ap4A）的α-手性硫代磷酸酯类似物；它们的酶促合成与降解

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

作者信息

Lazewska D, Guranowski A

机构信息

Katedra Biochemii, Akademia Rolnicza, Poznań, Poland.

出版信息

Nucleic Acids Res. 1990 Oct 25;18(20):6083-8. doi: 10.1093/nar/18.20.6083.

DOI:10.1093/nar/18.20.6083

PMID:2172926

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC332409/

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.

摘要

已在两种酶系统中对Ap4AαS的Sp和Rp非对映异构体的合成进行了表征，这两种酶系统分别是来自大肠杆菌的赖氨酰 - tRNA合成酶和来自酿酒酵母的Ap4Aα，β - 磷酸化酶。该合成酶能够使用（Sp）ATPαS和（Rp）ATPαS作为腺苷酸的受体，从而产生相应的Ap4A单硫类似物，即（Sp）Ap4AαS和（Rp）Ap4AαS。未形成二硫代磷酸类似物。Ap4A、（Sp）Ap4AαS和（Rp）Ap4AαS形成的相对合成酶速度为1:0.38:0.15，并且（Sp）ATPαS和（Rp）ATPαS的计算Km值分别为0.48和1.34 mM。酵母Ap4A磷酸化酶以腺苷5'-磷酸硫酸酯（APS）作为腺苷酸来源合成了（Sp）Ap4AαS和（Rp）Ap4AαS。腺苷酸被ATP的相应硫代类似物接受。在该系统中，Ap4A、（Sp）Ap4AαS和（Rp）Ap4AαS形成的相对速度为1:0.15:0.60。测试了Ap4A的两种异构硫代磷酸酯类似物作为以下特定Ap4A降解酶的底物：来自黄羽扇豆（Lupinus luteus）种子的（不对称）Ap4A水解酶（EC 3.6.1.17）将每种类似物水解为AMP和ATPαS的相应异构体；来自大肠杆菌的（对称）Ap4A水解酶（EC 3.6.1.41）产生ADP和ADPαS的相应非对映异构体；来自酿酒酵母的Ap4A磷酸化酶仅在未修饰的末端切割Rp异构体，产生ADP和（Rp）ATPαS，而Sp异构体被非特异性降解，产生ADP、（Sp）ADPαS、ATP和（Sp）ATPαS的混合物。对于所有Ap4A降解酶，Ap4AαS的Rp异构体似乎比其Sp对应物是更好的底物；这三种酶对Ap4AαS非对映异构体的立体选择性分别为51、6和2.5。给出了降解反应的基本动力学参数，并讨论了Ap4A代谢酶对在Ap4A - Pα处修饰的潜在底物的结构要求。