SMS ENZYME

Exploring Structure-Function Relationship of Enzymes at Single Molecule Level: Fluorescence and Force Spectroscopy

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 Obiettivo del progetto (Objective)

'The relation between structure and functions is a central topic in enzymology. I propose to study that question at the single molecule level employing a novel optical and force spectroscopy. The enzyme will be linked to a glass surface employing PEG-Biotin-Streptavidin interaction. The location of a single enzyme will be determined by a dye attached to it. The substrate of the reaction, while being non-fluorescent, will be chosen so that its enzymatic reaction product is fluorescent. The time span between fluorescence bursts reflecting the distribution of enzymatic active states will indicate the turnover rate of the enzyme. Contrary to the expectation that an enzyme is characterised by a well defined catalytic rate, a stretched exponential function has been reported to be necessary to fit the the probability distribution of the time span between bursts. I will study the effect of temperature on this stretched exponential behaviour for different enzymes. I will explore the enzymes labelled at two specific position by dyes which can act as donor and acceptor for resonance energy transfer (FRET) and the distance dependent FRET efficiency will offer an insight into the conformation fluctuation of the enzyme. In parallel to the optical observation, I will apply a mechanical tension on the enzymes thereby altering the distribution of its active states. In collaboration with the group of Dr. Marc Baaden, starting from the known enzymatic structure we will study numerically how the tension affects the enzymatic conformations. I will study how this tension alters its functions, via the changes in the distribution of turnover times. These studies will offer an insight into the relation between the structural changes of an enzyme and its measured catalytic activity.'

Introduzione (Teaser)

Life as we know is largely sustained by enzymes, which speed up vital metabolic processes. Studying enzymatic behaviour has implications for both health and disease.

Descrizione progetto (Article)

Enzymes are proteins that have the inherent capacity to catalyse specific chemical reactions. Through a specialised part of their three-dimensional structure, known as the active site, they interact with specific substrates to transform them into products.

To shed light on the relation between enzyme structure and function, studies at the single molecule level are required. Scientists on the EU-funded SMS ENZYME project proposed to address this need using optical and force spectroscopy. Their approach involved attaching a dye to the enzyme for localisation and ensuring that the enzymatic reaction product is fluorescent to study the distribution of enzymatic active states.

Contrary to the expectation that an enzyme is characterised by a well-defined catalytic rate, most studies report a stretched exponential function. To elucidate the conformational fluctuations of the enzymes, the consortium exploited the phenomenon of fluorescent resonance energy transfer. Furthermore, they applied mechanical tension to assess the distribution of enzyme active sites and study enzymatic conformation and function.

In practice, researchers implemented this approach for DNA-based reactions using DNA as an enzyme. They observed that when the DNA template was unstructured, it behaved as an ideal Michaelis-Menten enzyme. However when the DNA template was structured, the enzyme displayed non-exponential behaviour which resembles more closely the real enzymatic reactions. These findings indicated that switching between different conformations caused DNA to adopt a non-classical enzyme behaviour.

Taken together, these studies provide invaluable insight into the relation between the structural changes of an enzyme and its measured catalytic activity. This information has great implications for understanding vital biological processes but could also be extrapolated for the biotechnological utilisation of enzymes.