Reactions of the sarcoplasmic reticulum calcium adenosinetriphosphatase with adenosine 5'-triphosphate and Ca2+ that are not satisfactorily described by an E1-E2 model.
Reactions of the sarcoplasmic reticulum calcium adenosinetriphosphatase with adenosine 5'-triphosphate and Ca2+ that are not satisfactorily described by an E1-E2 model.
复制标题
E1-E2 模型未能令人满意地描述肌浆网钙三磷酸腺苷酶与 5-三磷酸腺苷和 Ca2 的反应。
DOI：
10.1021/bi00398a019
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发表时间：
1987
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：
Stahl,N;Jencks,WP
02254马萨诸塞州沃尔瑟姆布兰德大学生物化学研究生学系1987年4月1日收到修订稿件摘要：游离酶与饱和的三磷酸腺苷和50微米钙混合后，肌浆网钙三磷酸腺苷酶的磷酸化为一级反应，kh=70±7 S‘1；这是在相同条件下(pH 7.0，钙载囊泡，100 mM Kc1，5 mM镁2+，25℃)与钙预先孵育的酶的磷酸化速率常数E的三分之一。在0.25mMADP存在下，E与ATP和Ca~(2+)发生磷酸化，得到~50%的E~P-Ca~(2+)，其fcobsd=77 S“1，而不是正反向速率常数之和kobsi=k{+kt=140 S‘1，如果磷酸化是限速的，则有望接近平衡。这些结果表明：(1)Kh代表缓慢的构象变化，而不是磷酰化转移；(2)E和CE-Ca2的磷酸化遵循不同的途径。E与饱和的ATP和Ca~(2+)的磷酸化反应没有滞后，表明包括Ca~(2+)离子结合和磷酸转移在内的所有其他步骤的速率常数均为>；500 S‘1。用未标记的ATP或乙二醇双(d-氨基乙基醚)-Ar，Ar，7V/，7V-四乙酸(EGTA)进行的Chase实验表明，[-32]ATP和Ca~(2+)的解离速率常数与kb相近。在47 S‘1和24 S’1分别发生E*ATP-Ca~(2+)和E-ATP的解离。约20%的磷酸化发生在加入300微米三磷酸腺苷和50微米钙离子到酶后4.5毫秒的EGTA追逐之后。这表明，在构象变化(FCB)之前，钙离子从小泡外迅速与游离酶结合。在加入Ca~(2+)和追逐超标记三磷酸腺苷后，捕捉到的无Ca~(2+)·[-32]的比例在6.8µCa~(2+)处达到一半最大，Hill斜率=1.8。计算出E-ATP-Ca~(2+)对Ca~(2+)的解离常数为-2.2X~(-10)2(K_(0.5)=15µ)。当[Ca~(2+)]从50µm降至10 nm时，E~P-Ca~(2+)与1.1 mM ADP两相反应的慢相速率常数增加2.5倍，当Ca~(2+)浓度从1.7µm降至10 nm时，E~P-Ca~(2+)两相反应的速率常数几乎达到最大值。这表明，Ca~(2+)是从另一个物种AE-ATP-Ca~(2+)上解离出来的，AE-ATP-Ca~(2+)对Ca~(2+)具有很高的亲和力，并使Ca~(2+)与K<；45 S‘1解离。结论是，在大多数情况下，ATPase的稳态翻转是通过E-ATP-Ca~(2+)途径进行的，而不是通过CE-Ca~(2+)途径(或“E_1’Ca~(2+)”途径)。这导致11-17%的未磷酸化的酶在饱和[ATP]和[Ca~(2+)]时处于稳定状态，因为kb步骤部分是限速的。磷酸化的两条途径可以导致ATP的非线性Lineweaver-Burk曲线和磷酸酶水平的初始超调。肌浆网的钙ATPase(E)1与其底物ATP和Ca~(2+)的反应可以通过两条途径发生，具体取决于ATP和Ca~(2+)的浓度。方程1中的上一条途径是众所周知的钙首先结合并引起构象变化的途径。
Graduate Department of Biochemistry, Brandéis University, Waltham, Massachusetts 02254 Received April 1, 1987; Revised Manuscript Received July 7, 1987 abstract: Phosphorylation of the sarcoplasmic reticulum calcium ATPase, E, is first order with kh= 70±7 s'1 after free enzyme was mixed with saturating ATP and 50 µ Ca2+; this is one-third the rate constant of 220 s'1 for phosphorylation of enzyme preincubated with calcium, E-Ca2, after being mixed with ATP under the same conditions (pH 7.0, Ca2+-loaded vesicles, 100 mM KC1, 5 mM Mg2+, 25 C). Phosphorylation of E with ATP and Ca2+ in the presence of 0.25 mM ADP gives~ 50% E~ P-Ca2 with fcobsd= 77 s" 1, not the sum of the forward and reverse rate constants, kobsi= k {+ kT= 140 s'1, that is expected for approach to equilibrium if phosphorylation were rate limiting. These results show that (1) kh represents a slow conformational change, rather than phosphoryl transfer, and (2) different pathways are followed for the phosphorylation of E and of CE-Ca2. The absence of a lag for phosphorylation of E with saturating ATP and Ca2+ indicates that all other steps, including the binding of Ca2+ ions andphosphoryl transfer, have rate constants of> 500 s'1. Chase experiments with unlabeled ATP or with ethylene glycol bis (d-aminoethyl ether)-Ar, Ar, 7V/, 7V-tetraacetic acid (EGTA) show that therate constants for dissociation of [-32] ATP and Ca2+ are comparable to kb. Dissociation of ATP occurs at 47 s'1 from E* ATP-Ca2+ and at 24 s'1 from E-ATP. Approximately 20% phosphorylation occurs following an EGTA chase 4.5 ms after the addition of 300 µ ATP and 50 µ Ca2+ toenzyme. This shows that Ca2+ binds rapidly to the free enzyme, from outside the vesicle, before the conformational change (fcb). The fraction of Ca2+-free·[-32] that is trapped to givelabeled phosphoenzyme after the addition of Ca2+ and a chase of uiilabeled ATP is half-maximal at 6.8 µ Ca2+, with a Hill slope of= 1.8. The calculated dissociation constant for Ca2+ from E-ATP-Ca2 is-~ 2.2 X'10 2 (K0 5= 15 µ). The rate constant for the slowphase of the biphasic reaction of E~ P-Ca2 with 1.1 mM ADP increases 2.5-fold when [Ca2+] is decreased from 50 µ to 10 nM, with half-maximal increase at 1.7 µ Ca2+. This shows that Ca2+ is dissociating from a different species, aE-ATP-Ca2, that is active for catalysis of phosphoryl transfer, has a high affinity for Ca2+, and dissociates Ca2+ with k< 45 s'1. It is concluded that steady-state turnover of the ATPase under most conditions occurs through the E-ATP-Ca2 pathway, which has a relatively low affinity for Ca2+, notthe pathway through CE-Ca2 (or “E1'Ca2”). This results in 11-17% unphosphorylated enzyme in the steady state at saturating [ATP] and [Ca2+] because the kb step is partly rate limiting. The two pathways for phosphorylation can result in nonlinear Lineweaver-Burk plots for ATP and initial overshoots of phosphoenzyme levels.Reactions of the calcium ATPase (E) 1 of sarcoplasmic re-ticulum with its substrates ATP and Ca2+ can occur by two pathways, depending on the concentrations of ATP and Ca2+. The upper pathway in eq 1 is the well-known pathway in which calcium binds first and causes a conformational change before