The Control of Non-Chemical Steps in Enzyme Catalysis

Enzymes control the rate at which almost all chemistry of life occurs. They are also at the core of therapeutic intervention, industrial biotechnology, and synthetic biology, where biology is being utilised in entirely new ways. The manipulation of enzyme activity is a key element in each of these areas of research. Hence, understanding what determines the activity of enzymes is a high priority. Enzyme activity has been studied for many decades but important aspects of how they work have eluded the scientific approaches available at the time. Very recently, tools have been developed that allow the examination of enzymes at the resolution of almost every individual atom, telling us about their properties, how they interact and how they move. This opens up the opportunity to examine in far greater detail than has been possible to-date how enzymes behave at different stages of their reaction cycles. A proper understanding of all of these elements and their interplay is crucial to manipulating enzyme activity. In this study we will address three questions. Firstly we will examine why certain amino acids at specific positions in enzymes are the same in a wide variety of different organisms and yet, according to current models, have no role in how the enzymes work. Secondly, we will examine whether enzymes use tricks that sometimes occur when two proteins interact that make sure that the interaction is specific but neither strong nor long lasting. Thirdly, we will examine whether a behaviour found in some enzymes when they are in a resting phase are specifically present to ensure that the whole population of an enzyme is not too active unless the conditions are right. This scenario occurs when an enzyme does more than one job, and it is detrimental to be too fast doing one of the jobs. The overall context of the study is to develop better predictive models that improve the utilization of enzymes in therapeutic, industrial biotechnology and synthetic biology settings.

This application focuses on fundamental issues that are critical to the function of enzymes. Enzymes have to do much more than preferentially bind the transition state for the reaction to be effective. They have to bind and release the substrate(s), product(s) and any intermediate(s) with appropriate affinities and rates, otherwise those processes will become rate-limiting, and be severely detrimental to overall turnover. Using an archetypal phosphoryl transfer enzyme, we will utilise our ability to isolate the individual steps within catalysis to investigate how enzymes control the affinities and rates of substrate, product and intermediate binding. We will exploit the ever-evolving, sophisticated toolkit for the detailed examination of structure, electronics and dynamics within proteins, built around multi-dimensional heteronuclear NMR, X-ray crystallography, and microsecond MD simulations of complexes involved in phosphoryl transfer. These measurements will be combined with the detailed examination of the reaction cycle determined using state-of-the-art kinetics measurements. We have chosen beta-phosphoglucomutase 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 balanced. More specifically, we will address the three questions regarding fundamental properties that are evolved into enzymes to mitigate against substrate, product and intermediate release being seriously detrimental to function. The individual questions are: Are some surface residues conserved specifically to accelerate product release? Are some core residues conserved specifically to accelerate product release? Are some proline residues conserved specifically to regulate the level of activity?

We aim to resolve fundamental questions relating to how enzymes are equipped to release substrates and products while maintaining extremely high apparent affinities for the transition state associated with the chemical step in catalysis. This will lead to a step change in the ability of the community to understand more completely how enzymes function and how best to target strategies that modulate their function. These studies will not only provide a firmer foundation upon which to understand the roles of different components of enzymes in general, but also to accelerate progress in synthetic biology, biotechnology, and therapeutic development through understanding how to control affinities and rates of reactions in these ultra-complex systems.

The primary industrial beneficiaries of this work will be in the biotechnological and pharmaceutical sectors.
1. Biotechnological industries: by providing a firmer footing from which to control enzyme activity for industrial use. Tuning enzyme activity and affinity are often limiting factors in the efficiency of redesigned enzyme-based systems.
2. Pharmaceutical industry: by providing a better understanding of how enzymes control the binding of their natural ligands, thereby helping them to develop more specific, targeted drug treatments.
One of the primary driving forces behind developing the proposed research programme was discussions as part of our strategic alliance in biophysics with AstraZeneca. This area was identified as a key area where academic-led research can closely support aspirations within Pharma, but which lay beyond the scope of direct industrial funding. The general value of the programme to Pharma and Biotech also emerged from meetings with industrial advisors of the Krebs Institute Management Group (Sheffield), of which the PI is an executive member, and industrial partners of the CoEBP (Manchester), where the applicants play an active role. Many represented companies have a substantial interest in manipulating enzyme activity and stated that they would benefit considerably from an improved understanding of the mechanisms of control used by these proteins. They provided the strongest encouragement to address these fundamental issues from an academic perspective.

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.