On the Catalytic Mechanism of Human ATP Citrate Lyase
On the Catalytic Mechanism of Human ATP Citrate Lyase
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DOI：
10.1021/bi300611s
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发表时间：
2012-06-26
期刊：
影响因子：
2.9
通讯作者：
Meek, Thomas D.
中科院分区：
文献类型：
--
作者：
Fan, Fan;Williams, Howard J.;Meek, Thomas D.
三磷酸腺苷柠檬酸裂解酶(ACL)催化依赖于三磷酸腺苷的生物合成反应，由柠檬酸和辅酶A(CoA)合成乙酰辅酶A和草酰乙酸酯。用重组人ACL进行研究，以确定启动ACL反应的催化磷酸化的性质和所涉及的活性部位残基的身份。用焦碳酸二乙酯处理使ACL失活，表明活性部位组氨酸(即His760)的催化作用，它被认为在催化过程中形成磷酸组氨酸物种。用[Gamma-P-33]-ATP预稳态磷酸化ACL的pH依赖性揭示了一个pK(A)值接近7.5的可电离基团，该基团必须去质子化才能发生ACL的催化磷酸化。His760突变为丙氨酸导致ACL的生物合成反应失活，这与催化组氨酸的参与很好地一致。通过用[Gamma-O-18(4)]-ATP进行位置同位素交换，进一步研究了磷酸-ACL形成的本质。在没有柠檬酸和辅酶A的情况下，[γ-O-18(4)]-三磷酸腺苷的β、伽马桥到非桥的位置同位素交换速率达到最大值14 S(-1)。当加入柠檬酸盐时，这一比率降至5 S(-1)，当柠檬酸盐和辅酶A同时存在时，这一比率为10 S(-1)。快速的位置同位素交换率表明存在一种或多种催化相关、高度可逆的磷酸化中间体。在没有柠檬酸和辅酶A的情况下的稳态测量表明，野生型和H760A形式的ACL都产生了镁ADP，其合成反应的速率比k(CAT)低3个数量级。ACL的ATPase活性，以及在H760A突变体ACL中观察到的少量但显著的位置同位素交换率(类似于野生型的150倍)，共同表明ACL中存在第二次磷酸基转移，尽管是非生产性的。数学分析和计算机模拟表明，脱附速率接近7 S(-1)的速率是乙酸乙酯和草酰乙酸酯生物合成的限速步骤。
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 [gamma-P-33]-ATP revealed an ionizable group with a pK(a) value of similar to 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 [gamma-O-18(4)]-ATP. The beta,gamma-bridge to nonbridge positional isotope exchange rate of [gamma-O-18(4)]-ATP achieved its maximal rate of 14 s(-1) 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 (similar to 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 similar to 7 s(-1) was the rate-limiting step in the biosynthesis of AcCoA and oxaloacetate.