Institute for Chemical Biology & Drug Discovery, Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, United States.
Biochemistry. 2011 Dec 13;50(49):10678-86. doi: 10.1021/bi201199x. Epub 2011 Nov 17.
β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.
β-酮酰基-ACP 合酶（KAS）酶在脂肪酸生物合成途径中催化 Claisen 缩合反应。这些反应遵循乒乓机制，其中供体底物酰化活性位点半胱氨酸残基，然后酰基与丙二酰-ACP 受体底物缩合形成β-酮酰基-ACP。在起始 KASIII 酶中，供体底物是酰基辅酶 A，而在延伸的 KASI 和 KASII 酶中，供体是酰基-ACP。尽管大肠杆菌（ecFabH）中的 KASIII 酶是必需的，但结核分枝杆菌（mtFabH）中的相应酶不是必需的，这表明结核分枝杆菌（KasA 或 KasB，分别）中的 KASI 或 II 酶必须能够接受 CoA 供体底物。由于 KasA 是必需的，因此使用基于磷酸泛酰巯基乙胺、CoA、ACP 和 AcpM 肽模拟物的底物探索了这种 KASI 酶的底物特异性。这项分析已经扩展到大肠杆菌（ecFabB 和 ecFabF）中的 KASI 和 KASII 酶，我们表明，14 个残基的丙二酰磷酸泛酰巯基乙胺肽可以有效地替代丙二酰-ecACP 作为 ecFabF 反应的受体底物。虽然 ecFabF 能够在 CoA 是两种底物的载体时催化缩合反应，但 KASI 酶 ecFabB 和 KasA 绝对需要 ACP 底物作为酰基供体。只要满足此要求，受体载体底物的变化对 KASI 反应的 k(cat)/K(m)影响不大。对于 KASI 酶，我们提出 ecACP（AcpM）的结合导致构象变化，导致酶的开放形式，丙二酰受体底物结合于此。最后，当棕榈酰-CoA 是 KasA 反应的供体底物时观察到的底物抑制对 mtFabH 在分枝杆菌 FASII 途径中的重要性有影响。
