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.
Determination of Enzyme Kinetics Using Stopped-Flow Spectroscopy