Enzyme catalysis of nucleophilic attack of anions by anions

The present application aims to get to the heart of fundamental questions that are critical to developing our understanding of how enzymes work. Enzymes, which are normally proteins, control the rate at which almost all chemistry occurs in living systems. Understanding enzyme activity is a high priority - it is at the core of therapeutic intervention, industrial biotechnology, and synthetic biology. The controlled manipulation of enzyme activity is one of the key elements targeted in each of these areas of research. Enzyme activity has been studied for many decades and many paradigms have evolved but, very recently, tools have been developed that allow the testing of those paradigms with unprecedented levels of detail. We are now able to observe the structure, electronics and dynamics within enzymes at the level of individual atoms. A proper understanding of all of these elements and their interplay is crucial to manipulating enzyme activity, and with observation powers at this level of detail, many of traditional paradigms of enzymology are not surviving rigorous testing. In this study we will examine how enzymes handle chemistry between two entities with the same charge, how the enzyme is triggered to open and close, and how it avoids getting stuck in deep thermodynamic wells when dealing with high-energy reactions. These issues are fundamental to the activity of many enzymes and are not well understood. To achieve the study we will investigate the behaviour of an enzyme that moves phosphate groups between a carboxylate group and a phosphate group, and between two phosphate groups. Enzymes that move phosphate groups lie at the heart of every system in living organisms - in the storage, maintenance and expression of genetic information, in metabolism, communication, cell architecture, differentiation, and homeostasis. We will develop new models based on the behaviour an archetypal enzyme that delivers very high quality measurements, which can then be translated to other enzymes that are common targets in therapeutic, industrial biotechnology and synthetic biology programmes.

In order for an enzyme to be effective, it has to do much more than preferentially bind the transition state for the reaction. As a minimum, it has to bind and release the substrate(s) and product(s) with appropriate affinities and rates, and it has to set up the conformation(s) in which the chemical step occurs. Relating to all of these processes there are elements that are categorised under the heading of dynamics, but the picture of their involvement in catalysis is invariably clouded by the inability of the experiment to dissect which dynamics are associated with each element. Using an archetypal phosphoryl transfer enzyme, we have found ways to isolate key processes of catalysis. We aim to exploit this and the ever-evolving, sophisticated toolkit for the detailed examination of structure, electronics and dynamics within proteins to establish how this enzyme controls elements of catalysis: namely, how it brings together the attacking group and the target when they carry the same charge, how it controls domain opening and closing, and how it is designed to avoid detrimental, deep free energy traps. More specifically, the three questions that we will address are: How does an enzyme's active site mediate anionic attack on an anion? How does it initiate domain closure? How does it avoid making a prohibitively stable closed complex? Our approaches to answering the individual questions are built around multi-dimensional heteronuclear NMR, X-ray, chemical, QM and MM investigations of complexes of phosphoryl transfer enzymes. We have chosen human phosphoglycerate kinase as the principal study target on account of both its high tractability for the proposed study and preliminary data that have led to the proposal of new hypotheses about how catalysis is controlled.

Improving means with which the activity of enzymes can be modulated specifically and controllably is a primary goal of a spectrum of industry ranging from pharmaceuticals through agrochemicals to industrial biotechnology. The experience of the last 20 years has illustrated beyond any doubt that progress in all of these fields requires a combination of screening methods and more detailed, knowledge-driven methods, where the proposed research has its primary impact. Every biological system known has a reliance on the enzyme-mediated transfer of phosphate groups to or from biomolecules. Unsurprisingly, therefore, phosphoryl transfer enzymes have been a focus for a wide spectrum of the academic community and for almost every major company targeting biological systems. Despite very substantial investment and the importance of the targets, progress worldwide in the development of reagents that interfere with the activity of phosphoryl transfer enzymes has been patchy. One of the principal problems is that our understanding of the operational details of these enzymes is not nearly as sophisticated as is desirable to guide product development. We seek to make a step change in the level of understanding of the activity of phosphoryl transfer enzymes, which will provide a firmer foundation upon which to build a more comprehensive scientific understanding of catalysis by enzymes with obvious industrial and medicinal benefits. While the work that we propose is of a fundamental nature, the targets that are relevant to our studies include entities that have been identified as some of the most desirable targets for therapeutic intervention in cancer and in heart disease.

The proposed work packages focus on PGK. PGK deficiencies are an X-linked recessive trait associated with myopathy, hemolytic anemia, and mental disorders, and are currently untreatable. PGK is also necessary for in vivo activation of several antiretroviral drugs. Independent of its catalytic activity, PGK has a role in angiogenesis and tumour generation, and in cellular regulation through altering the stability of a subset of mRNAs. The understanding of these roles of PGK is far from complete and will benefit from the atomic level interrogation of PGK proposed here.

The work will be disseminated to as wide an audience as possible. The primary source of dissemination of the results derived from the proposed studies will be through publication in high impact scientific journals. We have a long track record in publishing our research findings in major international, non-specialist journals, aimed at a broad interest readership. In addition, we will continue to disseminate the work at international scientific conferences that are highly attended by both academic and industrial delegates. Furthermore, we will continue our excellent track record in depositing and supporting data in publically accessible biological databases.

Outside of these academic-focussed activities, we have been engaged in numerous direct activities involving industry and non-specialist audiences. Much of the proposed work stems from discussions with representatives of two major UK pharmaceutical companies and two leading biotechnology companies, all with substantial interests in phosphoryl transfer enzymes, and all of which would benefit considerably from an improved understanding of the activity and control of these enzymes. Hence, their encouragement to address these fundamental issues from an academic perspective.

Longer term should our efforts lead to improved therapeutic treatments or biotechnology processes it will increase UK industrial competitiveness and benefit society. However, all research and knowledge also benefits society, for example public communication/schools visit describing a new piece of research may be the inspiration for a career in science.