OBSERVATION OF MULTIPLE MYOSIN SUBFRAGMENT-1-ADP FLUOROBERYLLATE COMPLEXES BY F-19 NMR-SPECTROSCOPY
OBSERVATION OF MULTIPLE MYOSIN SUBFRAGMENT-1-ADP FLUOROBERYLLATE COMPLEXES BY F-19 NMR-SPECTROSCOPY
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DOI：
10.1021/bi00090a022
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发表时间：
1993-10-05
期刊：
影响因子：
2.9
通讯作者：
SYKES, BD
中科院分区：
文献类型：
--
作者：
HENRY, GD;MARUTA, S;SYKES, BD
氟铝酸盐和氟铍酸盐是肌球蛋白ATP酶活性的有效抑制剂。抑制需要ADP的存在，并且已经积累了许多证据表明四面体氟铝酸盐和氟铍酸根离子作为磷酸盐类似物，在通常由ATP的末端磷酸盐占据的位置中的活性位点处以高亲和力结合。S1-ADP-氟铝酸盐和S1-ADP-氟铍酸盐物质被认为类似于肌动球蛋白ATP酶循环中的动力学中间体。通过F-19 NMR表征S1-结合的氟铝酸盐是简单的;容易观察到被鉴定为AlF 4-的单一共振[Maruta，S.，亨利，G. D、赛克斯，B。D、& Ikebe，M（1993）J.Biol.Chem.268，7093-7100]。相反，发现结合的氟铍酸盐产生四个独立的峰：在-80和-83.5ppm处的低场对和在-101.5和-103ppm处的高场对，表明存在四种不同类型的S1-ADP-氟铍酸盐复合物。束缚共振的相对强度可以通过改变复合物形成过程中的F：Be比来改变。在氟标记的核苷酸衍生物3 '（2'）-O-（4-氟苯甲酰基）-ADP代替ADP的情况下获得的光谱的积分产生1.7-1.9：1的结合的氟化物与核苷酸的比率，表明主要结合的氟铍酸盐物质不像通常认为的那样是BeF 3-。结合的氟代铍酸盐对应于中性物种BeF_2（H_2O）_2和BeFOH（H_2O）_2以及带负电荷的物种[BeF_2OH.H_2O]-和[BeF_3-H_2O]-，但也讨论了其他可能性。
Fluoroaluminate and fluoroberyllate are potent inhibitors of the ATPase activity of myosin. Inhibition requires the presence of ADP, and much evidence has accumulated to suggest that the tetrahedral fluoroaluminate and fluoroberyllate ions act as phosphate analogues, binding with high affinity at the active site in the position normally occupied by the terminal phosphate of ATP. Both the S1-ADP-fluoroaluminate and the S1-ADP-fluoroberyllate species are thought to resemble kinetic intermediates in the actomyosin ATPase cycle. Characterization of S1-bound fluoroaluminate by F-19 NMR is straightforward; a single resonance identified as AlF4- is observed easily [Maruta, S., Henry, G. D., Sykes, B. D., & Ikebe, M (1993) J. Biol. Chem. 268, 7093-7100]. Bound fluoroberyllate, by contrast, was found to give rise to four separate peaks: a downfield pair at -80 and -83.5 ppm and an upfield pair at -101.5 and -103 ppm, suggesting the existence of four distinct types of S1-ADP-fluoroberyllate complex. The relative intensities of the bound resonances can be altered by changing the F:Be ratio during complex formation. Integration of a spectrum acquired in the presence of a fluorine-labeled nucleotide derivative, 3'(2')-O-(4-fluorobenzoyl)-ADP, in place of ADP yielded a bound fluoride to nucleotide ratio of 1.7-1.9 to 1, showing that the major bound fluoroberyllate species cannot be BeF3- as is usually thought. It is proposed that the bound fluoroberyllates correspond to the neutral species BeF2(H2O)2 and BeFOH(H2O)2 and the negatively charged species [BeF2OH.H2O]- and [BeF3-H2O]-, although other possibilities are discussed.