Understanding enzyme-catalysed phosphoryl transfer

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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.