Microsecond reaction kinetics and catalytic mechanism of bacterial cytochrome oxidases

Microsecond reaction kinetics and catalytic mechanism of bacterial cytochrome oxidases

Paulus, A. (TU Delft BT/Enzymology)

de Vries, S. (promotor)
Hagen, W.R. (promotor)

Delft University of Technology

2017-05-12

Fundamental biochemical research is of crucial importance for a complete and detailed understanding of what drives enzyme activity and how enzyme kinetic properties are optimized towards survival of the host organism. When cells fail to produce a fully functional enzyme, the organism’s ability to survive or thrive is impacted. In humans, for example, low levels or absence of lactase causes lactose intolerance, while decreased performance of the proton-pumping enzyme cytochrome aa3 oxidase in the mithochondrial electron transport chain in brain cells is linked to Alzheimer’s disease. Direct observation of all steps of the catalytic cycle of bacterial oxidoreductases is challenging, since a full turnover of these enzymes typically takes only ~1 ms. Through targeted mutagenesis of enzymes it is possible to create variants of an enzyme that can onlycatalyze part of the reaction, or that will perform the entire reaction, yet with different kinetics of the individual reaction steps, providing clues as to what drives or limits the enzymatic reaction. Hypotheses based on observations with mutated enzyme variants can be proven or disproven by studying the wildtype uncorrupted enzyme under mild conditions, minimizing artefacts introduced by working in vitro. The stopped-flow spectrophotometer is a valuable tool for kinetic analysis of enzyme reactions, but does not offer the time resolutionrequired to resolve early pre-steady state kinetics. The microsecond freeze-hyperquenching setup (MHQ), on the other hand, is able to create ‘snapshot’ samples of enzymes during catalytic turnover at reaction times down to 74 μs. The quenched samples can be subjected to further analysis by UV-vis or EPR spectroscopy. This thesis decribes the kinetic study of three bacterial oxidoreductases and makes the comparison between the catalytic mechanismsof oxygen reduction (and proton pumping) of each of the three enzymes.

https://doi.org/10.4233/uuid:28e0fdfa-72f2-4feb-9fd0-fcc807bb3593

978-94-028-0630-4

Institutional Repository

doctoral thesis

© 2017 A. Paulus