NMR studies of the AMP-binding site and mechanism of adenylate kinase.
NMR studies of the AMP-binding site and mechanism of adenylate kinase.
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腺苷酸激酶的 AMP 结合位点和机制的 NMR 研究。
DOI：
10.1021/bi00380a024
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Mildvan,AS
中科院分区：
文献类型：
--
作者：
Fry,DC;Kuby,SA;Mildvan,AS
1986年10月23日摘要：核磁共振以前曾被用来确定酶结合MgATP的构象，并定位腺苷酸激酶上MgATP结合位点[Fry，DC，Kuby，S.一、& Mildvan，A. S.（1985）Biochemistry 24，4680-4694]。为了确定构象和位置的其他基板，AMP，距离已被测量从Cr 3 + AMPPCP，一个线性竞争性抑制剂相对于MgATP，六个质子和磷原子的腺苷酸激酶上的AMP，与顺磁探针-7/方法。用含时核奥弗豪泽效应（NOE）测量了酶结合AMP上的5个质子间距离。这些距离被用来确定结合AMP的构象以及它相对于金属-ATP的位置。酶结合AMP具有高的反糖基扭转角（x= 110±10）、3 '-内、2'-外核糖折叠（δ = 105±10）以及围绕C4 '-C5'键（δ = 180±10）和CS '-CU'键（f3= 170±20）的gauche-反式取向。Cr ~（3+）与AMP中磷的距离为5.9±0.3 A，表明反应配位距离约为3 A，这与磷酰基转移的缔合SN ~ 2机理一致。10个分子间NOE，从酶的质子到AMP的质子，被检测到，表明至少有三个疏水性氨基酸的接近结合AMP。这些限制，连同AMP的构象和底物间的距离，被用来定位到腺苷酸激酶的X射线结构的AMP。发现AMP结合位点靠近（< 4 A）Leu-116、Arg-171、瓦尔-173、瓦尔-182和Leu-190;已经发现所有这些残基在肌肉型兔、小牛、人、猪中是不变的[Kuby，S.一、帕尔米耶里河H、Frischat，A.，Fischer，AH，Wu，LH，Maland，L.，& Manship，M.（1984）Biochemistry 23，2393-2399]和鸡腺苷酸激酶[Kishi，F.，Maruyama，M.，Tanizawa，Y.，& Nakazawa，A.（1986）J.Biol.Chem.261，2942-2945]，腺苷酸激酶在可逆反应MgATP+ AMP → MgADP+ ADP中催化磷酰基的转移该酶含有两个不同的底物结合位点，其中一个结合MgATP和MgADP，另一个对未复合的AMP和ADP特异（Hamada和Kuby，1978; Rhoads和Lowenstein，1968; Nageswara Rao等，1978年）。酶上这些结合位点的位置一直是本实验室感兴趣的问题。我们最近已经确定了MgATP在兔肌肉腺苷酸激酶上的结合位点（Fry等人，1985）通过在完整的酶和一种酶的溶液中进行广泛的NMR研究，
Revised Manuscript Received October 23, 1986 abstract: NMR has previously been used todetermine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, DC, Kuby, S. A., & Mildvan, A. S.(1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+ AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-7/method. Time-dependent nuclear Overhauser effects (NOEs) havebeen used to measure five interproton distances on enzyme-bound AMP. These distances were used todetermine the conformationof bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anfi'-glycosyl torsional angle (x= 110±10), a 3'-endo, 2'-exo ribose pucker (8= 105±10), and gauche-trans orientations about the C4'-C5'bond (7= 180±10) and the CS'-CU'bond (f3= 170±20). The distance from Cr3+ to the phosphorus of AMP is 5.9±0.3 A, indicating a reaction coordinate distance of~ 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (< 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, AH, Wu, LH, Maland, L., & Manship, M.(1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y., & Nakazawa, A.(1986) J. Biol. Chem. 261, 2942-2945],^^. denylate kinase catalyzes the transfer of a phosphoryl group in the reversible reaction MgATP+ AMP^ MgADP+ ADPThe enzyme contains two distinct substrate binding sites, one of which binds MgATP and MgADP and the other of which is specific for uncomplexed AMP and ADP (Hamada & Kuby, 1978; Rhoads & Lowenstein, 1968; Nageswara Rao et al., 1978). The location of these binding sites on the enzyme has been a problem of ongoing interest to this laboratory. We have recently determined the binding site of MgATP on rabbit muscle adenylate kinase (Fry et al., 1985) through an extensive NMR study in solution of both the intact enzyme and a