The binding of ATP and Mg2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum follows a random mechanism.
The binding of ATP and Mg2+ to the calcium adenosinetriphosphatase of sarcoplasmic reticulum follows a random mechanism.
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ATP 和 Mg2 与肌浆网钙腺苷三磷酸酶的结合遵循随机机制。
DOI：
10.1021/bi00077a016
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Jencks,WP
中科院分区：
文献类型：
--
作者：
Reinstein,J;Jencks,WP
1993年3月17日收到的修订版摘要：肌浆网腺苷三磷酸钙酶（CaATPase）的酶形式cE-Ca 2在钙存在下稳定，具有催化Mg 2+离子的结合位点，在25 ℃、pH 7.0和100 mM KCl下解离常数为0.94±0.15 mM。这比在类似条件下报道的游离酶E（8.8mM）的值小= 10倍[Punzengruber，C.，普拉格河，Kolassa，N.，Winkler，F.，& Suko，J.（1978）Eur. J.Biochem.92，349-359]，这种差异表明催化离子和转运离子的位点在不存在ATP的情况下相互作用。在Ca ~（2+）和Mg ~（2+）孵育的酶中加入ATP和EDTA，磷酸化酶的生成率为61%。将未标记的ATP和Mg 2+加入到已经与3.5 µ游离Ca 2+和标记的ATP孵育的酶中，得到39%的标记的磷酸酶。这表明ATP和Mg 2+与cE-Ca 2的结合遵循随机机制。ATP和Mg 2+从cE-Ca 2-ATP-Mg解离的速率常数不同：fcdiss（ATP）= 120 s-1和fcdiss（Mg 2+）= 60 s-1。这表明Mg 2+和ATP可以独立地结合和解离;它们不必作为Mg· ATP复合物与CE缔合或解离。不含钙的酶在活性位点结合不含金属的ATP，解离常数为44±4 µ，knss= 130±7 s-1，计算的结合速率常数为3 × 106 M-1 s-1。与[-32]孵育的无钙酶在与未标记的ATP、Mg 2+和Ca 2+追逐时产生38%的标记的磷酸酶。Mg 2+浓度的增加没有增加E32 P的形成量。这表明Mg 2+和ATP与游离E的结合也遵循随机机制。Mg 2+离子不被ATP所掩埋，而ATP也不被Mg 2+离子所掩埋。游离E与Mg ~（2+）和ATP共同孵育引起构象变化，激活酶磷酸化，使ATP解离速率常数从/cd_（ss）= 120 s ~（-1）降低到k_（ss）= 47 s~*。肌浆网（SR）的钙腺苷三磷酸酶（CaATPase）1负责将Ca ~（2+）从肌肉组织转运到SR内腔。在Mg 2+作为催化离子的存在下，ATP水解为ADP和无机磷酸盐的自由能提供了逆着电化学梯度泵送Ca 2+的能量（哈塞尔巴赫& Makinose 1961，1963; Ebashi & Lipmann，1962）。方案一所示的运输周期（Makinose，1973）包括两个钙离子与SR细胞质侧的ATP的结合（Yamamoto & Tonumura，1967），将ATP的末端磷酸基团转移至天冬氨酸351（艾伦和绿色，1976）形成酸稳定的磷酸酶（Degani & Boyer，1973），并且Ca 2+解离到SR的内腔，这允许磷酸酐键的水解以完成循环。磷酸酶的形成和分解伴随着反转发生，这导致在整个反应中观察到的构型保留（Webb & Trentham，1981）。
Revised Manuscript Received March 17, 1993 abstract: The enzyme form of the calcium adenosinetriphosphatase of sarcoplasmic reticulum (CaATPase) that is stable in the presence of calcium, cE-Ca2, has a binding site for the catalytic Mg2+ ion with a dissociation constant of 0.94±0.15 mM at 25 C, pH 7.0, and 100 mM KC1. This is= 10 times smaller than that reported for the free enzyme, E,(8.8 mM) under similar conditions [Punzengruber, C., Prager, R., Kolassa, N., Winkler, F., & Suko, J.(1978) Eur. J. Biochem. 92, 349-359], This difference shows that the sites for the catalytic and the transported ions interact in the absence of ATP. The addition of ATP and EDTA to enzyme that had been incubatedwith Ca2+ and Mg2+ resulted in the formation of 61% phosphoenzyme. The addition of unlabeled ATP and Mg2+ to enzyme that had been incubated with 3.5 µ free Ca2+ and labeled ATP gave 39% labeled phosphoenzyme. This shows that the binding of ATP and Mg2+ to cE-Ca2 follows a random mechanism. The rate constants for dissociation of ATP and Mg2+ from cE-Ca2-ATP-Mg are different:/cdiss (ATP)= 120 s-1 and fcdiss (Mg2+)= 60 s_1. This shows that Mg2+ and ATP can bind and dissociate independently; they do not have to associate or dissociate from CE as a Mg· ATP complex. Calcium-free enzyme binds metal-free ATP at the active site with a dissociation constant of 44±4 µ, knss= 130±7 s~', and a calculated associationrate constant of 3 X 106 M" 1 s-1. Calcium-free enzyme that was incubated with [-32] gave 38% labeled phosphoenzyme when chased with unlabeled ATP, Mg2+, and Ca2+. An increase of the Mg2+ concentration did not increase the amount of E32P formed. This shows that the binding of Mg2+ and ATP to free E also follows a random mechanism. The Mg2+ ion is not buried under ATP, and ATP is notunder a Mg2+ ion. Incubation of free E with Mg2+ and ATP causes a conformational change that activates the enzyme for phosphorylation and decreases the rate constant for the dissociation of ATP from/cd¡ ss= 120 s™ 1 to k¿ iss= 47 s~*.The calcium adenosinetriphosphatase (CaATPase) 1 from sarcoplasmic reticulum (SR) transports Ca2+ ions from muscle tissue into the lumen of theSR. The energy for pumping Ca2+ against an electrochemical gradient is provided by the free energy of hydrolysis ofATP to ADP and inorganic phosphate in the presence of Mg2+ as the catalytic ion (Hasselbach & Makinose 1961, 1963; Ebashi & Lipmann, 1962). The transport cycle shown in Scheme I (Makinose, 1973) includes the bindingof two calcium ions and ATP from the cytosolic side of the SR (Yamamoto & Tonumura, 1967), transfer of the terminal phosphate group of ATP to aspartate 351 (Allen & Green, 1976) to form acid-stablephosphoenzyme (Degani & Boyer, 1973), and dissociation of Ca2+ to the lumen of the SR, which allows hydrolysis of the phosphate anhydride bond to complete the cycle. Theformation and breakdown of the phosphoenzyme occur with inversion, which leads to the retention of configuration that is observed for the overall reaction (Webb & Trentham, 1981).
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无机磷酸盐对钙腺苷三磷酸酶的磷酸化：高镁离子浓度下的可逆抑制。
影响因子：
2.9
作者：
Loomis,CR;Martin,DW;McCaslin,DR;Tanford,C
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DOI：
10.1016/s0021-9258(19)40236-6
发表时间：
1990
期刊：
The Journal of biological chemistry
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作者：
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DOI：
--
发表时间：
1990
期刊：
The Journal of biological chemistry
影响因子：
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作者：
Fujimori,T;Jencks,WP
通讯作者：
Jencks,WP
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影响因子：
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作者：
S. Highsmith;M. Head
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M. Head
DOI：
--
发表时间：
1975
期刊：
影响因子：
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作者：
A. F. Hegarty;W. Jencks
通讯作者：
W. Jencks