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Investigate enzymatic mechanisms

Investigate enzymatic mechanisms using stopped-flow

Stopped-Flow spectroscopy is one of the primary techniques for studying enzyme kinetics. It has been used for decades to the elucidate the details of enzymatic reactions and is widely used in many areas of modern biochemical research including drug discovery and development, and synthetic biology.

Enzyme mechanisms often have multiple intermediate steps, typically involving substrate binding, product release and intermediate catalytic steps. These steps are all as fast or faster than the steady state turnover rate of the enzyme, so they can only be probed with techniques that can measure reactions on millisecond to second time scales. Using stopped-flow spectroscopy to look at the details of the individual steps of an enzymatic reaction in an initial catalytic turnover, it becomes possible to separate and understand the individual reaction steps, such as substrate and product binding or reaction intermediates, identify any rate-limiting steps and understand the reaction mechanism.

Enzymes and substrates usually contain chromophores that change their physical properties throughout the various phases of an enzymatic reaction. Using Stopped-Flow spectroscopy, we use highly sensitive detection and signal processing to measure changes in spectroscopic signal (absorbance or fluorescence) to monitor directly monitor the reaction kinetics.

The data measured can be fit to various kinetic models to give us rate constants that can be in turn used to derive binding affinities, catalytic efficiencies, and infer conformational state changes.
Pre-steady state kinetic analysis can allow us to identify individual chemical intermediates such as the state of the cofactors during complex multistep reaction mechanisms associated with redox-active proteins. Examples include flavoproteins or metal complexes such as heme proteins. We can also study the effect of inhibitors, ligands, and other macromolecules on enzymatic activity.





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SX 20 Enzymes Teaser

Determination of Enzyme Kinetics Using Stopped-Flow Spectroscopy

This study describes the use of an SX20 spectrometer to measure the presteady state kinetics of a well-studied enzymatic reaction: hydrolysis of p-Nitrophenyl acetate catalysed by α-chymotrypsin. Kinetic parameters such as rate constants and the Michaelis-Menten constant are evaluated.

Since it was first studied over 50 years ago, extensive research has established the detailed mechanism of this reaction.

SF3 Teaser

Shining Light on Non-Chemical Steps in Protein Catalysis

Stopped-flow experiments using Applied Photophysics SX20 and Chirascan V100 SF.3 systems allow monitoring of different spectral properties including fluorescence, absorbance, and even far-and near-UV CD.With these tools. This study outlines the transition between fully folded and locally unfolded states of Tsa1, in its sulfenic acid form CP-SOH, is not in rapid equilibrium as previously hypothesised, but is almost irreversible (kFF~0s-1)and in direct competition with the hyperoxidation step.

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