Development of carbonic anhydrase mimics that target the inefficiencies of rubisco by increasing local CO2 concentration in situ
File(s)
Author(s)
Rains, Jon George David
Type
Thesis
Abstract
It is predicted that the global population will be over 9.8 billion people by 2050. In order
to meet this rise in population, an increase of at least 50% of crop yields will need to be achieved by this date. Increasing photosynthetic efficiency remains one of the few routes left for substantial increases in crop yields. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the
enzyme responsible for fixating carbon dioxide from the atmosphere, is one of the major bottlenecks of photosynthesis, due to its slow catalytic rate and low specificity for CO2 resulting in the competing photorespiration reaction pathway. Some organisms have evolved carbon
concentrating mechanisms (CCM’s) to increase photosynthetic efficiency, by increasing the local CO2 concentration around Rubisco via utilisation of a β-Carbonic Anhydrase (CA). This project
aims to chemically mimic the role of β-CA, through the use of small molecule metal-ligand complexes based on that of the previously known mimic ZnL1S, and to test their ability to improve Rubisco’s activity.
Prior to the development of the β-CA mimics, a thorough understanding of the mechanism of both the hydration and dehydration reactions as catalysed by ZnL1S, the fastest known water soluble CA mimic, was desired. For the first time, via stopped-flow, the dehydration profile of the fastest small-molecule CA mimic, ZnL1S, was characterised. Furthermore, the hydration reaction was studied at 25 oC across a large range of catalyst and substrate concentrations in the most comprehensive study of a CA mimic to date. Most significantly, an improvement in the
favourability of the rate-limiting step was achieved by lowering the substrate concentration, thus reducing the bicarbonate inhibition of the catalyst. Consequently, an increase in the mimic’s kcat for hydration and dehydration was observed, resulting in the largest rate constants of any small molecule CA mimic reported to date. Furthermore, a CA mimic demonstrated, for the first time, similar rates of dehydration and hydration at physiological pH’s under certain substrate
concentrations. The first experimental validation of the rate-limiting bicarbonate-release step, as predicted theoretically for ZnL1S, was achieved through NMR experiments, inhibitor effects on mimic kinetics and a study into the effect of the metal centre. Subsequently, the first IC50 and ki values for CA mimic inhibitors of CO2 hydration were generated. Finally, enzyme-like kcat / Km values were obtained for ZnL1S allowing for the mimic’s true potential as a catalyst to be compared to the natural CA enzyme. Notably there is only a difference of 2.5 orders of magnitude,
the closest of any CA mimic reported in the literature.
Subsequently, a suite of existing and novel water insoluble CA mimics was synthesised
based on modified structures of ZnL1S. The mimics synthesised contained a varying number of sulfur atoms and coordinating arms, and therefore were more structurally similar to that of the desired β-CA found in CCM’s in plants and cyanobacteria. There was a general trend of increasing preference for the dehydration reaction over the hydration reaction with increasing number of sulfur atoms and 3 coordinating arms, although the best performing mimic contained 2 sulfur containing and 1 nitrogen containing arms, much like β-CA itself. Furthermore, with high substrate concentration, dehydration was found to be faster than hydration for some of the mimics. Mimics with a greater preference for the dehydration reaction also showed a greater positive effect on Rubisco activity in vitro. Consequently, the effect of the mimics on spinach growth in vivo was tested. To track the distribution of the mimics in vivo, the synthesis of fluorescent water-soluble versions of the mimics was attempted and is still ongoing. Future work will focus on improving the bioavailability of the mimics for optimal performance in vivo.
to meet this rise in population, an increase of at least 50% of crop yields will need to be achieved by this date. Increasing photosynthetic efficiency remains one of the few routes left for substantial increases in crop yields. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), the
enzyme responsible for fixating carbon dioxide from the atmosphere, is one of the major bottlenecks of photosynthesis, due to its slow catalytic rate and low specificity for CO2 resulting in the competing photorespiration reaction pathway. Some organisms have evolved carbon
concentrating mechanisms (CCM’s) to increase photosynthetic efficiency, by increasing the local CO2 concentration around Rubisco via utilisation of a β-Carbonic Anhydrase (CA). This project
aims to chemically mimic the role of β-CA, through the use of small molecule metal-ligand complexes based on that of the previously known mimic ZnL1S, and to test their ability to improve Rubisco’s activity.
Prior to the development of the β-CA mimics, a thorough understanding of the mechanism of both the hydration and dehydration reactions as catalysed by ZnL1S, the fastest known water soluble CA mimic, was desired. For the first time, via stopped-flow, the dehydration profile of the fastest small-molecule CA mimic, ZnL1S, was characterised. Furthermore, the hydration reaction was studied at 25 oC across a large range of catalyst and substrate concentrations in the most comprehensive study of a CA mimic to date. Most significantly, an improvement in the
favourability of the rate-limiting step was achieved by lowering the substrate concentration, thus reducing the bicarbonate inhibition of the catalyst. Consequently, an increase in the mimic’s kcat for hydration and dehydration was observed, resulting in the largest rate constants of any small molecule CA mimic reported to date. Furthermore, a CA mimic demonstrated, for the first time, similar rates of dehydration and hydration at physiological pH’s under certain substrate
concentrations. The first experimental validation of the rate-limiting bicarbonate-release step, as predicted theoretically for ZnL1S, was achieved through NMR experiments, inhibitor effects on mimic kinetics and a study into the effect of the metal centre. Subsequently, the first IC50 and ki values for CA mimic inhibitors of CO2 hydration were generated. Finally, enzyme-like kcat / Km values were obtained for ZnL1S allowing for the mimic’s true potential as a catalyst to be compared to the natural CA enzyme. Notably there is only a difference of 2.5 orders of magnitude,
the closest of any CA mimic reported in the literature.
Subsequently, a suite of existing and novel water insoluble CA mimics was synthesised
based on modified structures of ZnL1S. The mimics synthesised contained a varying number of sulfur atoms and coordinating arms, and therefore were more structurally similar to that of the desired β-CA found in CCM’s in plants and cyanobacteria. There was a general trend of increasing preference for the dehydration reaction over the hydration reaction with increasing number of sulfur atoms and 3 coordinating arms, although the best performing mimic contained 2 sulfur containing and 1 nitrogen containing arms, much like β-CA itself. Furthermore, with high substrate concentration, dehydration was found to be faster than hydration for some of the mimics. Mimics with a greater preference for the dehydration reaction also showed a greater positive effect on Rubisco activity in vitro. Consequently, the effect of the mimics on spinach growth in vivo was tested. To track the distribution of the mimics in vivo, the synthesis of fluorescent water-soluble versions of the mimics was attempted and is still ongoing. Future work will focus on improving the bioavailability of the mimics for optimal performance in vivo.
Version
Open Access
Date Issued
2019-04
Date Awarded
2019-10
Copyright Statement
Creative Commons Attribution NonCommercial NoDerivatives Licence
Advisor
Barter, Laura
Long, Nicholas
Woscholski, Rudiger
Sponsor
Engineering and Physical Sciences Research Council
Grant Number
CHTP_G01437
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)