Understanding enzyme-catalysed phosphoryl transfer

生物体化学最显著的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提供极其稳定的骨架，DNA和RNA，另一方面提供蛋白质活性的暂时和瞬时调节，主要受蛋白激酶和蛋白磷酸酶的控制。同时，通过操纵磷酸及其酯的酸酐，特别是三磷酸腺苷，磷酸酯也被用来在整个生命系统中产生、分配和应用能量。解决磷酸单酯和双酯显著的化学稳定性与其易操纵性之间的矛盾的办法在于所谓的磷酰基转移酶的催化能力，它可以快速地建立和断裂P-O-C和P-O-P键，这导致了一些迄今已知的最大的酶速率加速。尽管磷酸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控方式只被理解在相对初级的水平。其中一个主要原因是，由于相关物种的寿命如此之短，很难在催化的短暂时刻观察到酶。解开这一难题最有意义的实验方法是将一种化学物质插入酶中，这种化学物质与短暂的种群物种非常相似，但在更长的时间内是稳定的。我们发现了一种新型的无机物种，它比以前发现的任何磷酸转移酶都能更好地发挥这一作用，借此镁和氟在酶的活性部位结合，从而极好地模拟正在转移的磷酸基团。这种酶的精致陷阱，就像在转移磷酸基团的过程中捕捉到它一样，使我们能够测量酶如何能够提供使磷酰基转移以对生物系统有用的速度发生所需的巨大稳定性。我们将使用我们也开发的实验和计算方法的组合来分析捕获的酶，这是一个协同和跨学科的研究计划。这一认识的发展将使我们能够指导调节磷酸基转移酶活性的化合物的演变，这些化合物是从心脏病和癌症到作物保护等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规定的目标，即朝着对生物过程更多的数学和物理理解的方向发展。

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.