Transient tunnels that assemble and disassemble to facilitate passage of unstable intermediates in enzymes containing multiple reaction centers are controlled by allosteric cues. Using the 140-kDa purine biosynthetic enzyme PurL as a model system and a combination of biochemical and x-ray crystallographic studies, we show that long-distance communication between ~25-Å distal active sites is initiated by an allosteric switch, residing in a conserved catalytic loop, adjacent to the synthetase active site. Further, combinatory experiments seeded from molecular dynamics simulations help to delineate transient states that bring out the central role of nonfunctional adaptor domains. We show that carefully orchestrated conformational changes, facilitated by interplay of dynamic interactions at the allosteric switch and adaptor-domain interface, control reactivity and concomitant formation of the ammonia tunnel. This study asserts that substrate channeling is modulated by allosteric hotspots that alter protein energy landscape, thereby allowing the protein to adopt transient conformations paramount to function.

组装和拆卸以促进不稳定的中间体通过包含多个反应中心的酶的瞬态通道受变构线索控制。使用140 kDa嘌呤生物合成酶PurL作为模型系统，并结合生化和X射线晶体学研究，我们发现〜25Å远端活性位点之间的长距离通讯是由一个变构开关引发的，该开关位于一个保守的催化环，与合成酶活性位点相邻。此外，从分子动力学模拟中获得的组合实验有助于描述瞬态，从而发挥非功能性衔接子域的核心作用。我们表明，精心设计的构象变化，通过变构开关和衔接子-域界面的动态相互作用的相互作用而得以促进，控制氨通道的反应性和随之形成。这项研究断言，底物通道受到变构热点的调节，这些变构热点改变了蛋白质的能量分布，从而使蛋白质能够采用对功能至关重要的瞬时构象。