Investigations of kinase substrate specificity with aqua Rh(III) complexes of adenosine 5'-triphosphate.
Investigations of kinase substrate specificity with aqua Rh(III) complexes of adenosine 5'-triphosphate.
复制标题
使用 5-三磷酸腺苷水性 Rh(III) 复合物研究激酶底物特异性。
DOI：
10.1021/bi00060a032
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Dunaway-Mariano,D
中科院分区：
文献类型：
--
作者：
Lu,Z;Shorter,AL;Dunaway-Mariano,D
摘要：本文报道了/ 3,7 -双齿Rh (H20) 4ATP和/ 8,7 -三齿Rh (H20) 3ATP立体异构体对激酶家族选定成员的底物活性和结合亲和力。己糖激酶和甘油激酶对A/ 3,7 -双齿Rh (H20) 4ATP异构体具有特异性，而腺苷酸激酶对18,7 -双齿Rh (H20) 4ATP异构体具有特异性催化作用。丙酮酸激酶识别(8,7 -双齿Rh (H20) 4ATP异构体和j8-P，外显式p a，/ 3,7 -三齿Rh (H20) 3ATP异构体作为底物，催化磷酸化替代底物乙醇酸。各自产物的31P核磁共振分析表明，在催化之前或之后都没有磷酰配体交换。肌酸激酶被发现对a8 - p，外显式，8,7 -三齿Rh (H20) 3ATP异构体具有特异性。Rh (H20) nATP异构体在己糖激酶和腺苷酸激酶的优先结合中被观察到，但在甘油激酶、果糖-6-磷酸激酶、肌酸激酶、精氨酸激酶或醋酸激酶中没有被观察到。Cleland和Mildvan的实验室首次使用配体交换速率慢的金属离子构建稳定的金属-核苷酸复合物，作为酶活性位点和机制的探针（Foster & Mildvan, 1972; DePamphilis & Cleland, 1973）。近年来制备的co111和Crmnucleotide复合物在酶动力学机制、底物结构、底物和辅因子结合位点以及金属离子催化等方面的研究中得到了应用[相关综述，参见Cleland and Mildvan（1979）、Cleland（1982, 1985）和Dunaway-Mariano(1985)]。然而，Cr （III）和Co-(III)金属离子的固有性质限制了它们作为酶活性位点探针的使用。具体来说，在Com-ATP1系列中，7-单齿、/ 3,7 -双齿和a，/ 3,7 -三齿异构体的全谱是可用的，但只能作为相应的五胺、四胺和三胺配合物（Cornelius等，1977；Speckhard等，1986;Knight, 1984）。这些复合物的Co111胺中心对天然激酶底物Mgn (H20) " ATP的Mgnaqua中心的模拟效果很差。这反映在观察到的Com （NH4）”ATP复合物与激酶的弱结合和相对较少的证明底物活性的例子上。与氧化还原不稳定的水系ComATP复合物不同，稳定的水系CrmATP复合物可以
Revised Manuscript Received December 3, 1992 abstract: In this paper the substrate activities and bindingaffinities of the stereoisomers of the/3, 7-bidentate Rh (H20) 4ATP and a, 8, 7-tridentate Rh (H20) 3ATP complexes toward selected members of the kinase family of enzymes are reported. Hexokinase and glycerokinase were found to be specific for the A/3, 7-bidentate Rh (H20) 4ATP isomer as substrate while adenylate kinase was found to specifically catalyze the reaction of the 18, 7-bidentate Rh (H20) 4ATP isomer. Pyruvate kinase recognized both the(8, 7-bidentate Rh (H20) 4ATP isomer and the j8-P, exo-P a,/3, 7-tridentate Rh (H20) 3ATP isomer as substrates in the catalyzed phosphorylation of the alternate substrate, glycolate. 31P NMR analysis of the respective product complexesshowed that-P phosphoryl ligand exchange had not preceded or followed catalysis. Creatine kinase was found to be specific for the A 8-P, exo-P a, 8, 7-tridentate Rh (H20) 3ATP isomer. Discrimination of the Rh (H20) nATP isomers viapreferential binding of the substrate-active isomer was observed for hexokinaseand adenylate kinase but not for glycerokinase, fructose-6-phosphatekinase, creatine kinase, arginine kinase, or acetate kinase.The use of metal ions having slow ligand exchange rates to construct stable metal-nucleotide complexes for use as probes of enzyme active sites and mechanisms was initiated inthe laboratories of Cleland and Mildvan (Foster & Mildvan, 1972; DePamphilis & Cleland, 1973). The Co111-and Crmnucleotide complexes that have been prepared in recent years have found application in the study of enzyme kinetic mechanisms, substrate structure, substrate and cofactor binding sites, and metal ion catalysis [for reviews, see Cleland and Mildvan (1979), Cleland (1982, 1985), and Dunaway-Mariano (1985)]. Nevertheless, the intrinsic properties of the Cr (III) and Co-(III) metal ions have placed certain restrictions on their use as enzyme active site probes. Specifically, within the Com-ATP1 series, the full spectrum of 7-monodentate,/3, 7-bidentate, and a,/3, 7-tridentate isomers is available but only as the corresponding pentaamine, tetraamine, and triamine complexes (Cornelius et al., 1977; Speckhard et al., 1986; Knight, 1984). The Co111 amine center of these complexes is a poor mimic for the Mgnaqua center of the natural kinase substrate, Mgn (H20)„ATP. This is reflected by theobserved weak binding of the Com (NH4)„ATP complexes to kinases and by the relatively fewexamples of demonstrated substrate activity. Unlike the aqua ComATP complexes, which are redox unstable, stable aqua CrmATP complexes can be