Department of Chemistry, Connecticut College, New London, Connecticut 06320, USA.
J Am Chem Soc. 2011 Jul 27;133(29):11088-91. doi: 10.1021/ja2041496. Epub 2011 Jul 1.
According to the domain alternation mechanism and crystal structure evidence, the acyl-CoA synthetases, one of three subgroups of a superfamily of adenylating enzymes, catalyze adenylate- and thioester-forming half-reactions in two different conformations. The enzymes accomplish this by presenting two active sites through an ~140° rotation of the C-domain. The second half-reaction catalyzed by another subgroup, the beetle luciferases, is a mechanistically dissimilar oxidative process that produces bioluminescence. We have demonstrated that a firefly luciferase variant containing cysteine residues at positions 108 and 447 can be intramolecularly cross-linked by 1,2-bis(maleimido)ethane, trapping the enzyme in a C-domain-rotated conformation previously undocumented in the available luciferase crystal structures. The cross-linked luciferase cannot adenylate luciferin but is nearly fully capable of bioluminescence with synthetic luciferyl adenylate because it retains the ability to carry out the oxidative half-reaction. The cross-linked luciferase is apparently trapped in a conformation similar to those adopted by acyl-CoA synthetases as they convert acyl adenylates into the corresponding CoA thioesters.
根据结构域转换机制和晶体结构证据，酰基辅酶 A 合成酶是腺苷酸化酶超家族的三个亚组之一，通过 C 结构域约 140°的旋转呈现两个活性位点，催化腺嘌呤和硫酯形成的半反应。另一个亚组的另一种半反应——甲虫荧光素酶，则是一种在机制上不同的氧化过程，产生生物发光。我们已经证明，一种含有位置 108 和 447 的半胱氨酸残基的萤火虫荧光素酶变体可以通过 1,2-双（马来酰亚胺基）乙烷进行分子内交联，将酶固定在以前在可用的荧光素酶晶体结构中未记录的 C 结构域旋转构象中。交联的荧光素酶不能使荧光素腺苷酸化，但由于它保留了进行氧化半反应的能力，几乎完全能够与合成的荧光基腺苷酸进行生物发光。交联的荧光素酶显然被困在类似于酰基辅酶 A 合成酶在将酰基腺苷酸转化为相应的 CoA 硫酯时所采用的构象中。
