Enzyme assays and kinetics

Beyond this limit the enzyme is saturated with substrate and the reaction rate ceases to increase. The reaction catalysed by an enzyme uses exactly the same reactants and produces exactly the same products as the uncatalysed reaction. Like other catalystsenzymes do not alter the position of equilibrium between substrates and products.

Enzyme assays and kinetics

Enzymes act on molecules, referred to as substrates, to form products. Enzyme kinetic parameters are determined via assays that directly or indirectly measure changes in substrate or product concentration over time. This video will cover the basic principles of enzyme kinetics including rate equations and kinetic models.

The concepts governing enzyme assays are also discussed, followed by a typical colorimetric assay.

Enzyme assays and kinetics

Enzymes are biochemical catalysts that are essential for life. Enzyme assays are used to study the kinetic properties of enzymatic reactions, elucidating the catalytic effects of enzymes. This video will cover enzyme kinetics and assays, go over a general procedure, and show some applications.

Enzymes are proteins or protein-like molecules that act on a reactant molecule, referred to as the substrate. Enzymes reduce the activation energy of biochemical reactions. This allows reactions to occur at faster rates with lower energy requirements. Enzymatic reactions can be broken up into three elementary components.

The first is the formation of the enzyme-substrate complex, formed by the binding of the substrate to the enzyme active site. The complex can decompose into its original constituents. This is the second elementary reaction. Alternatively, the Enzyme assays and kinetics can form the product and recover the enzyme, the third elementary reaction.

The kinetics of an elementary reaction is given by the elementary rate law equation. Rate law equations give the rate in terms of the concentration of the reactants and a rate constant.

Each of the elementary reactions has an individual rate law equation, with its own rate constant. These equations can be distilled down to a kinetic model known as the Michaelis-Menten equation. This gives the reaction rate in terms of the substrate concentration; which can be experimentally determined.

Some general trends for enzyme reactions can be identified using the Michaelis-Menten equation. At high substrate concentration, a saturation point is reached, called Vmax.

Here, the rate is limited by the total enzyme concentration, and the number of substrate molecules an enzyme converts into product per given time, also known as kcat. In Michaelis-Menten kinetics kcat is one of the two constants that govern reaction rate. The other constant, KM, is known as the affinity constant.

KM is also equivalent to the concentration where the reaction rate is equivalent to one-half Vmax. An enzyme with a higher affinity will have a lower KM and reach Vmax faster, while an enzyme with lower affinity will have a higher KM and take longer to reach Vmax.

Knowing kcat and KM allows for enzymes to be compared. To do this we use a ratio called enzyme efficiency. Higher kcat and lower KM result in higher efficiencies, while lower kcat and higher KM results in lower.

The factors used to elucidate enzyme kinetics must be determined experimentally. These assays are typically performed by mixing an enzyme and substrate solution in a controlled environment. Observations are made by measuring the changes in concentration of the substrate, product, or byproducts with respect to time.

Assay components

The change in concentration over time is used to determine the reaction rate. In order to determine the kinetics, rate data must be obtained at multiple concentrations.

Enzyme assays and kinetics

If a plot of the inverse initial rate vs. The slope and intercept of the line allow for the determination of the kinetic parameters KM and Vmax, which can then be used to calculate kcat and the enzyme efficiency. Now that the principles of enzyme kinetics have been discussed, let's look at how a typical enzyme assay is performed.

In this procedure a colorimetric assay is demonstrated.

Basics of Enzymatic Assays for HTS - Assay Guidance Manual - NCBI Bookshelf

The first step is to generate a standard curve, which will correlate absorbance with protein concentration. Solutions of known concentration are prepared along with a control sample. A developer solution that reacts with the target protein is added to produce a colored compound.

Absorbance is measured and plotted against concentration to generate the standard curve.Thankfully, this is the 21st century, and almost all kinetic assays are performed on temperature-controlled specs and allow you to set the temperature to your desired degree—25 °C, 30 °C, 37 °C, or whatever your temperature of interest may be.

When setting up an enzyme assay to study inhibition, it is worthwhile to use enzyme kinetics to measure the Michaelis constant (K m) for the enzyme's substrate, or for each of the substrates if it. Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.

Enzyme kinetics graph showing rate of reaction as a function of substrate concentration for normal enzyme, enzyme with a competitive inhibitor, and enzyme with a noncompetitive inhibitor.

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For the competitive inhibitor, Vmax is the same as for the normal enzyme, but Km is larger. Enzyme assays have many applications in enzyme kinetics. Understanding the rates of reactions can help determine the mechanism that the reaction follows (a single-substrate or multiple-substrate mechanism).

Figure 1 below demonstrates how enzymes change a reaction's mechanism by lowering the activation energy. Working with Enzymes: Part I -The Simple Kinetic Spectrophotometric Assay By Christopher Dieni At the end of my last article, I provided some practical tips and tricks for working with enzymes at the bench.

Working with Enzymes: Part I -The Simple Kinetic Spectrophotometric Assay - Bitesize Bio