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On the catalytic mechanism of human ATP citrate lyase

机译：人ATP柠檬酸裂解酶的催化机理

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ATP citrate lyase (ACL) catalyzes an ATP-dependent biosynthetic reaction which produces acetyl-coenzyme A and oxaloacetate from citrate and coenzyme A (CoA). Studies were performed with recombinant human ACL to ascertain the nature of the catalytic phosphorylation that initiates the ACL reaction and the identity of the active site residues involved. Inactivation of ACL by treatment with diethylpyrocarbonate suggested the catalytic role of an active site histidine (i.e., His760), which was proposed to form a phosphohistidine species during catalysis. The pH-dependence of the pre-steady-state phosphorylation of ACL with [γ- ~(33)P]-ATP revealed an ionizable group with a pK _a value of ～7.5, which must be unprotonated for the catalytic phosphorylation of ACL to occur. Mutagenesis of His760 to an alanine results in inactivation of the biosynthetic reaction of ACL, in good agreement with the involvement of a catalytic histidine. The nature of the formation of the phospho-ACL was further investigated by positional isotope exchange using [γ- ~(18)O _4]-ATP. The β,γ-bridge to nonbridge positional isotope exchange rate of [γ- ~(18)O _4]-ATP achieved its maximal rate of 14 s ~(-1) in the absence of citrate and CoA. This rate decreased to 5 s ~(-1) when citrate was added, and was found to be 10 s ~(-1) when both citrate and CoA were present. The rapid positional isotope exchange rates indicated the presence of one or more catalytically relevant, highly reversible phosphorylated intermediates. Steady-state measurements in the absence of citrate and CoA showed that MgADP was produced by both wild type and H760A forms of ACL, with rates at three magnitudes lower than that of k _(cat) for the full biosynthetic reaction. The ATPase activity of ACL, along with the small yet significant positional isotope exchange rate observed in H760A mutant ACL (～150 fold less than wild type), collectively suggested the presence of a second, albeit unproductive, phosphoryl transfer in ACL. Mathematical analysis and computational simulation suggested that the desorption of MgADP at a rate of ～7 s ~(-1) was the rate-limiting step in the biosynthesis of AcCoA and oxaloacetate.
机译：ATP柠檬酸裂解酶（ACL）催化ATP依赖的生物合成反应，该反应从柠檬酸和辅酶A（CoA）产生乙酰辅酶A和草酰乙酸。用重组人ACL进行了研究，以确定启动ACL反应的催化磷酸化的性质以及所涉及的活性位点残基的身份。通过用焦碳酸二乙酯处理使ACL失活表明了活性位点组氨酸（即His760）的催化作用，该组据建议在催化过程中形成磷酸组氨酸。 ACL的[γ-〜（33）P] -ATP的稳态磷酸化的pH依赖性显示其电离基团的pK _a值为〜7.5，对于ACL催化磷酸化为发生。 His760突变为丙氨酸会导致ACL的生物合成反应失活，这与催化组氨酸的参与非常吻合。通过使用[γ-〜（18）O _4] -ATP进行位置同位素交换，进一步研究了磷酸-ACL的形成性质。在不存在柠檬酸盐和CoA的情况下，[γ-〜（18）O _4] -ATP的β，γ桥到非桥位置同位素交换速率达到其最大速率14 s〜（-1）。当添加柠檬酸盐时，该速率降低至5 s〜（-1），而当同时存在柠檬酸盐和CoA时，该速率降至10 s〜（-1）。快速的位置同位素交换速率表明存在一种或多种催化相关的，高度可逆的磷酸化中间体。在没有柠檬酸盐和CoA的情况下进行稳态测量表明，MgADP是由野生型和H760A形式的ACL产生的，其速率比完全生物合成反应的k_（cat）低三个数量级。 ACL的ATP酶活性，以及​​在H760A突变型ACL中观察到的小而显着的位置同位素交换速率（比野生型低约150倍），共同表明ACL中存在第二个（尽管无用的）磷酰基转移。数学分析和计算模拟表明，MgADP的解吸速率约为7 s〜（-1），是AcCoA和草酰乙酸生物合成的限速步骤。

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