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Content archived on 2024-06-18

A study of the mechanistic basis for catalytic promiscuity in the alkaline phosphatase superfamily

Objective

Enzymes are the catalysts which allow important chemical reactions to occur on biologically relevant time-scales, and the current understanding of enzymes assumes that each enzyme is specifically tailored for a single reaction. Enzyme promiscuity refers to an enzyme’s ability to catalyze chemical transformations other than its native reactions. The identification of an increasing number of promiscuous enzymes challenges the classical “one enzyme, one activity” dogma.The notion that a single enzyme is capable of efficiently catalyzing a number of different reactions, which proceed through different transition states, is challenging to our current comprehension of enzymes. Although enzyme promiscuity is now a well documented phenomenon, the mechanistic principles underlying this behaviour are not well understood. This proposal aims to study catalytic promiscuity in the alkaline phosphatase superfamily, a class of phosphate and sulphate ester cleaving enzymes which are known to be prone to promiscuous behaviour. Specifically, linear free energy relationships will be used to characterize the bond-making and bond-breaking processes in phosphate/sulfate ester cleavage, and how the enzyme is able to stabilize these events. We will also use site directed mutagenesis and a high throughput selection system based on microdroplets to probe the contributions of individual amino acid residues to the catalytic mechanism and study enzyme evolution. Of particular interest is the question of how one enzyme active site is able to efficiently catalyze several thermodynamically demanding reactions which pass through transition states with vastly different properties under non-enzymatic conditions. In addition to enhancing our basic understanding of enzyme function and evolution, the mechanistic insight gleaned from this study will be instrumental in the directed evolution of enzymes with enhanced activity for specific uses in the biotech industry.

Topic(s)

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Call for proposal

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FP7-PEOPLE-2011-IIF
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Funding Scheme

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MC-IIF - International Incoming Fellowships (IIF)

Coordinator

THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
EU contribution
€ 200 371,80
Address
TRINITY LANE THE OLD SCHOOLS
CB2 1TN CAMBRIDGE
United Kingdom

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Region
East of England East Anglia Cambridgeshire CC
Activity type
Higher or Secondary Education Establishments
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Total cost

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