Sanders C R, Tian G C, Tsai M D
Department of Chemistry, Ohio State University, Columbus 43210.
Biochemistry. 1989 Nov 14;28(23):9028-43. doi: 10.1021/bi00449a011.
Adenylyl (beta,gamma-methylene)diphosphonic acid (AMPPCP) labeled with deuterium at the adenine ring ([8-2H]AMPPCP) and at the beta,gamma-methylene group (AMPPCD2P), as well as adenosine 5'-monophosphate labeled at the adenine ring ([8-2H]AMP), was synthesized and used for deuterium nuclear magnetic resonance (NMR) determination of effective correlation times (tau c) of the free nucleotide and the complexes with adenylate kinase (AK). Extensive and rigorous control experiments and theoretical analysis were performed to justify the validity of the experimental approaches, particularly the fast exchange condition, and the reliability of the tau c values obtained. For the free nucleotide, the results suggest that the phosphonate group of free AMPPCP possesses appreciable local mobility relative to the adenine ring and that complexation with Mg2+ greatly reduced such a local mobility. For the complexes with AK, effective tau c values of 7, 15, 28, 28, and 27 ns were obtained for AMPPCD2P, MgAMPPCD2P, [8-2H]AMPPCP, Mg[8-2H]AMPPCP, and [8-2H]AMP, respectively. These results suggest that the adenine ring of substrates is rigidly bound in all cases, that the phosphonate chain of AMPPCP possesses considerable local mobility, and that Mg2+ reduces such local mobility but does not totally immobilize it. The local dynamics of the analogues bound to AK was correlated with local binding energies for the binding of MgAMPPCP and MgATP to AK estimated from the binding studies by proton NMR and other techniques, in conjunction with the binding theory of Jencks [Jencks, W. P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4046-4050]. The results suggest that no general correlation exists between the local rigidity of portions of a bound substrate and the corresponding (ground state) local binding energy contributed by these portions. In particular, the adenosine moiety contributes little to the binding energy despite the fact that the adenine ring is rigidly bound; the triphosphate (PPPi) moiety behaves oppositely; Mg2+ immobilizes the triphosphate chain but does not enhance binding. Finally, isomers of the substitution-inert beta,gamma-bidentate Cr(III) complexes of adenosine 5'-triphosphate (CrATP) were used to probe two unresolved catalytic problems implicitly related to the local mobility of the phosphonate chain of AMPPCP in the AK-MgAMPPCP complex. The first problem concerns the result of electron paramagnetic resonance (EPR) studies that (Rp)- but not (Sp)-[beta-17O]ATP caused a line broadening in the Mn(II) EPR spectrum of the AK-MnATP complex [Kalbitzer, H. R., Marquetant, R., Connolly, B. A., & Goody, R. S. (1983) Eur. J. Biochem. 133, 221-227].(ABSTRACT TRUNCATED AT 400 WORDS)
腺嘌呤环上标记有氘（[8-2H]AMPPCP）以及β,γ-亚甲基基团上标记有氘（AMPPCD2P）的腺苷酰（β,γ-亚甲基）二磷酸（AMPPCP），还有腺嘌呤环上标记有氘（[8-2H]AMP）的腺苷5'-单磷酸，被合成出来并用于通过氘核磁共振（NMR）测定游离核苷酸以及与腺苷酸激酶（AK）形成的复合物的有效相关时间（τc）。进行了广泛且严格的对照实验和理论分析，以证明实验方法的有效性，特别是快速交换条件，以及所获得的τc值的可靠性。对于游离核苷酸，结果表明游离AMPPCP的膦酸酯基团相对于腺嘌呤环具有明显的局部流动性，并且与Mg2+络合大大降低了这种局部流动性。对于与AK形成的复合物，分别获得了AMPPCD2P、MgAMPPCD2P、[8-2H]AMPPCP、Mg[8-2H]AMPPCP和[8-2H]AMP的有效τc值为7、15、28、28和27纳秒。这些结果表明，底物的腺嘌呤环在所有情况下都紧密结合，AMPPCP的膦酸酯链具有相当大的局部流动性，并且Mg2+降低了这种局部流动性，但并未使其完全固定。结合质子NMR和其他技术的结合研究以及Jencks的结合理论[Jencks, W. P. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4046 - 4050]，将与AK结合的类似物的局部动力学与MgAMPPCP和MgATP与AK结合的局部结合能相关联。结果表明，结合底物部分的局部刚性与这些部分贡献的相应（基态）局部结合能之间不存在普遍的相关性。特别是，尽管腺嘌呤环紧密结合，但腺苷部分对结合能的贡献很小；三磷酸（PPPi）部分则相反；Mg2+使三磷酸链固定，但并未增强结合。最后，腺苷5'-三磷酸（CrATP）的取代惰性β,γ-双齿Cr(III)络合物的异构体被用于探究两个未解决的催化问题，这两个问题与AK - MgAMPPCP复合物中AMPPCP膦酸酯链的局部流动性隐含相关。第一个问题涉及电子顺磁共振（EPR）研究的结果，即（Rp） - 而不是（Sp） - [β-17O]ATP导致AK - MnATP复合物的Mn(II) EPR谱线加宽[Kalbitzer, H. R., Marquetant, R., Connolly, B. A., & Goody, R. S. (1983) Eur. J. Biochem. 133, 221 - 227]。（摘要截断于400字）
