Understanding enzyme-catalysed phosphoryl transfer

生物体化学的最显著特征之一是磷酸酯的进化发展，一方面为编码遗传信息的生物聚合物、DNA和RNA提供极其稳定的骨架，另一方面提供蛋白质活性的时间和瞬时调节，主要在蛋白激酶和蛋白磷酸酶的控制下。与此同时，磷酸酯也用于通过操纵磷酸及其酯的酸酐，特别是三磷酸腺苷，在整个生命系统中产生、分配和应用能量。磷酸单酯和磷酸二酯的显著化学稳定性与其容易操作之间的矛盾的解决方案在于所谓的磷酰基转移酶的催化能力，以快速形成和破坏P-O-C和P-O-P键，这引起了一些迄今为止确定的最大的酶促速率加速。尽管磷酰基转移酶在生物系统中处于中心地位，但催化能力的来源及其调节方式仅在相对初级的水平上被理解。其中一个主要原因是，由于相关物种的寿命很短，因此很难在催化的短暂时刻观察酶。解开这个难题的最有价值的实验方法是将一种化学物质插入酶中，这种化学物质提供了短暂的种群物种的近似模拟，但在更长的时间内是稳定的。我们已经发现了一种新型的无机物质，它比以前鉴定的任何磷酰基转移酶都能更好地发挥这一作用，镁和氟化物联合收割机在酶的活性部位结合，形成了一种极好的磷酸基团转移模拟物。酶的这种精巧的陷阱，就像在转移磷酸基团的过程中捕获它一样，使我们能够测量酶如何能够赋予使磷酰基转移以对生物系统有用的速率发生所需的巨大稳定性。我们将使用我们还开发的实验和计算方法相结合，在协同和跨学科的研究计划中分析捕获的酶。这种理解的发展将使我们能够指导调节磷酰基转移酶活性的化合物的演变，磷酰基转移酶是从心脏病和癌症到作物保护的广泛重要过程中的治疗和生物技术干预的目标。这项工作符合BBSRC的既定目标，即对生物过程进行更多的数学和物理理解。

One of the most remarkable features of the chemistry of living organisms is the evolutionary development of phosphate esters to provide on one hand the extremely stable backbone for biopolymers that encode genetic information, DNA and RNA, while on the other hand providing the temporal and transient regulation of protein activity, largely under the control of protein kinases and protein phosphatases. In the meantime, phosphate esters are also utilised for the generation, distribution, and application of energy throughout the living systems by the manipulation of anhydrides of phosphoric acid and its esters, notably adenosine triphosphate. The solution to the paradox between the remarkable chemical stability of phosphate mono- and di-esters and their facile manipulation lays in the catalytic power of so-called phosphoryl transfer enzymes to make and break P-O-C and P-O-P bonds rapidly, which gives rise to some of the largest enzymatic rate accelerations yet identified. Despite this central position for phosphoryl transfer enzymes in biological systems, the source of catalytic power and how it is regulated is understood only at a relatively rudimentary level. One of the principal reasons for this is that it is difficult to observe the enzymes in the fleeting moments of catalysis, since the lifetimes of the relevant species are so short. The most informative experimental approaches to unravelling this conundrum are where a chemical that provides a close mimic of the fleeting populated species, but which is stable for far more extended period, is inserted into the enzyme. We have discovered a new type of inorganic species that performs this role far better than any previously identified for phosphoryl transfer enzymes, whereby magnesium and fluoride combine in the active site of the enzyme to make an excellent mimic of a phosphate group being transferred. This exquisite trap of the enzyme, which catches it as if in the act of transferring a phosphate group, enables us to measure how the enzyme is able to impart the enormous stabilisation required to make phosphoryl transfer occur at a rate that is useful for biological systems. We will analyse the trapped enzyme using a combination of experimental and computational methods that we have also developed, in a synergistic and interdisciplinary research programme. The development of this understanding will enable us to guide the evolution of compounds that modulate the activity of phosphoryl transfer enzymes, which are targets for therapeutic and biotechnological intervention in a broad range of important processes from heart disease and cancer to crop protection. The work matches the stated aims of BBSRC in moving towards a more mathematical and physical understanding of biological processes.