Objective
The detailed mechanism of the oxidation of the plant hormone - indole-3-acetate - by molecular dioxygen catalysed by plant peroxidases and arachidonic acid oxidation by molecular dioxygen catalysed by mammalian prostaglandin H synthase (PGHS) will be investigated by means of anaerobic pre-steady-state kinetics, HPLC analysis of the reaction products and electrochemical kinetics. Organic hydroperoxides will be isolated and their reactivity towards a number of heme-containing peroxidases will be investigated. In order to understand the relationship between the cyclooxygenase and peroxidase sites in prostaglandin H synthase, comparisons will be made with an artificial bienzyme system comprising a Mn-porphyrin-substituted PGHS which lacks peroxidase activity coupled to an exogenous plant peroxidase.
The research activities will include
- Preparation of highly purified native, heme-substituted and recombinant enzymes: PGHS and Mn-porphyrin-substituted PGHS (Mn-PGHS), native peroxidases from tobacco, alfalfa, cucumber, wild-type and mutant recombinant horseradish peroxidase (HRP) forms overexpressed in E.coli;
- HPLC studies on IAA oxidation catalysed by plant peroxidases, their Mn-analogues and recombinant variant forms of HRP and arachidonic acid oxidation catalysed by PGHS and a bienzyme system (Mn-PGHS and HRP), isolation and characterisation of skatole hydroperoxide and prostaglandin G2 and studies of their reactivity toward plant and fungal peroxidases;
- Electrochemical and spectrophotometric steady-state kinetics of the artificial bienzyme system (Mn-PGHS and HRP), kinetic simulations and a comparison with native PGHS kinetics;
- Anaerobic stopped-flow studies on PGHS and plant peroxidases and the development of a comprehensive kinetic model;
- Studies on the mechanism of enzyme inactivation during oxygenation catalysis; Isolation and characterization of plant biologically active compounds affecting Prostaglandin H synthase and regulating prostaglandin biosynthesis enzyme site-specific inhibition and the development of stabilisation procedures to minimise free radical induced inactivation.
This project is expected to provide new insights into the catalytic mechanisms of two physiologically important oxygenase reactions: plant hormone indole-3-acetic acid oxidation by plant peroxidases and arachidonic acid oxidation by PGHS. The artificial bienzyme system utilising horseradish peroxidase and Mn-porphyrin-substituted PGHS will help define the relationship between the oxygenase and peroxidase sites in PGHS. This fundamental research in combination with the more applied inactivation studies will allow the development of new approaches to stabilise functioning PGHS in homogenous solution and on an electrode surface. These enzyme systems have potential for commercial exploitation in drug synthesis and in the development of biosensors for the analysis of metabolites and drug delivery control
Topic(s)
Call for proposal
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NR4 7UH Norwich
United Kingdom