Kurz L C, Nakra T, Stein R, Plungkhen W, Riley M, Hsu F, Drysdale G R
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1998 Jul 7;37(27):9724-37. doi: 10.1021/bi980325g.
This work reports the relative importance of the interactions provided by three catalytic residues to individual steps in the mechanism of citrate synthase. When the side chains of any of the residues (H320, D375, and H274) are mutated, the data indicate that they are involved in the stabilization of one or more of the transition/intermediate states in the multistep citrate synthase reaction. H320 forms a hydrogen bond with the carbonyl of oxaloacetate and the alcohols of the citryl-coenzyme A and citrate products. Enzymes substituted at H320 (Q, G, N, and R) have reaction profiles for which the condensation reaction is cleanly rate determining. None of these mutants can activate the carbonyl of oxaloacetate by polarization. All these mutants catalyze the necessary proton transfer from the methyl group of acetyl-coenzyme A only poorly, a process which occurs in a structurally separate site. Furthermore, all H320 mutants hydrolyze the citryl-coenzyme A intermediate significantly more slowly than does the wild-type. D375 is the base removing the proton of acetyl-coenzyme A. D375E and D375G have greatly diminished ability to catalyze proton transfer from acetyl-CoA. The D375 mutants polarize the oxaloacetate carbonyl as well as wild-type. For D375E, the hydrolysis of citryl-CoA is rate determining. D375G, having no side chain capable of acid-base chemistry in either the condensation or hydrolysis reactions is nearly completely devoid of activity in any of the reactions catalyzed by the wild-type. H274 hydrogen bonds to the carbonyl of acetyl-coenzyme A but also forms the back wall of the oxaloacetate-binding site. H274G cannot properly activate either oxaloacetate or acetyl-coenzyme A, and the condensation reaction is overwhelmingly rate determining. Nonetheless, hydrolysis of the intermediate is impaired. All the enzymes except H320R and H274G show kinetic cooperativity with CitCoA as substrate, indicating changes in the subunit interactions with these latter two mutants. The energetics of citrate synthase are surprisingly tightly coupled. All changes affect more than one step in the catalytic cycle. Within the condensation reaction, the intermediate of proton transfer must occupy a shallow well between transition states close in free energy so that perturbations of one have substantial effects on that of the other.
本研究报告了柠檬酸合酶机制中三个催化残基所提供的相互作用对各个步骤的相对重要性。当任何一个残基（H320、D375和H274）的侧链发生突变时，数据表明它们参与了柠檬酸合酶多步反应中一个或多个过渡/中间状态的稳定。H320与草酰乙酸的羰基以及柠檬酰辅酶A和柠檬酸产物的醇形成氢键。在H320处被取代（Q、G、N和R）的酶具有这样的反应曲线，即缩合反应是明确的速率决定步骤。这些突变体中没有一个能够通过极化作用激活草酰乙酸的羰基。所有这些突变体仅能很差地催化从乙酰辅酶A甲基进行的必要质子转移，该过程发生在一个结构上独立的位点。此外，所有H320突变体水解柠檬酰辅酶A中间体的速度明显比野生型慢得多。D375是去除乙酰辅酶A质子的碱。D375E和D375G催化从乙酰辅酶A进行质子转移的能力大大降低。D375突变体使草酰乙酸羰基极化的程度与野生型相同。对于D375E，柠檬酰辅酶A的水解是速率决定步骤。D375G在缩合或水解反应中都没有能够进行酸碱化学作用的侧链，在野生型催化的任何反应中几乎完全没有活性。H274与乙酰辅酶A的羰基形成氢键，但也构成了草酰乙酸结合位点的后壁。H274G不能正确激活草酰乙酸或乙酰辅酶A，并且缩合反应是压倒性的速率决定步骤。尽管如此，中间体的水解受到损害。除了H320R和H274G之外的所有酶都以CitCoA作为底物表现出动力学协同性，这表明这后两个突变体在亚基相互作用方面发生了变化。柠檬酸合酶的能量学惊人地紧密耦合。所有变化都会影响催化循环中的多个步骤。在缩合反应中，质子转移的中间体必须占据自由能相近的过渡态之间的一个浅阱，这样对其中一个的扰动会对另一个产生实质性影响。
