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Enzyme kinetic assay

Enzyme kinetics is the study of enzyme mechanisms through determination of reaction rates under varied conditions. The rate of a reaction is dependent on several factors including the concentration of the substrate and the enzyme, temperature, pH and presence of inhibitors.

At the top is an illustration of the Michaelis-Menten saturation curve. On the x-axis is substrate concentrations and on the y-axis is reaction rate. A Michaelis-Menten curve is fitted to the plot as a curve that increases fast and then plateaus shortly after. The data is marked with bullets and a line between them is drawn in blue. The graph shows that as the substrate concentration increases, so does the reaction rate. The reaction rate increase is rapid at the beginning of the reaction, but as the substrate concentration increases further, the reaction rate slows down and the curve starts plateauing. At this point, the reaction approaches its maximum velocity, which is marked on the graph as V max. Half of V max is also marked on the graph, where the reaction has met 50% of its maximum velocity. A third variable on the graph is the k m which is equal to the substrate concentration where the reaction rate is one half times V max. At the bottom is another illustration of a graph that has time on the x-axis and the formation of product on the y-axis. The curve reflects the general enzymatic reaction, which is shown as the change in concentration over time. The change is a result of the formation of the product created from the substrate. As time increases, so do the product in a linear fashion until the curve starts to plateau as a result of a decreasing reaction rate

Figure 1: Top: Michaelis-Menten saturation curve. Bottom: Progress curve of a general enzymatic reaction.

Performing kinetic assays

In a kinetic assay, the goal is to model the reaction rate, V, concerning substrate concentration, ([S]), illustrated in Figure 1. Measuring rates at various substrate concentrations is essential, generating progress curves showing product formation over time (also shown in Figure 1). Initially, the reaction rate remains constant but decreases as the substrate depletes, reaching a plateau. To account for changing substrate levels during the reaction, it's common to measure initial rates (V0) and plot them against substrate concentration. The initial rate is obtained from the linear segment of the progress curve at the reaction's outset.

The simplest model of V as a function of substrate concentration is the Michaelis-Menten equation. Reactions where the Michaelis-Menten equation can be applied, show an increase in the reaction rate when the substrate concentration is increased; however, this increase is diminished as the rate approaches the maximum velocity, Vmax as seen in Figure 1. The Michaelis-Menten equation will be further described on the following pages [1]. V max as seen in Figure 1. The Michaelis-Menten equation will be further described on the following pages.

Factors affecting the reaction rate

Temperature and pH play crucial roles in determining the reaction rate of enzymes. Enzymes exhibit an optimum pH, which relies on their unique composition. This dependence arises from the amino acid side chains' properties; certain side chains must be either protonated or deprotonated for the enzyme to function effectively. The pH of the solution and the side chain's pKa determine this protonation status. For example, histidine has a pKa of 6.0, rendering it mostly protonated at pH<6.0 and mostly deprotonated at pH>6.0.

Notably, the environment can alter the pKa of side chains, differing from that of free amino acids. Temperature also impacts the reaction rate: higher temperatures generally accelerate the rate until a critical point where the enzyme begins to denature, causing a subsequent decrease in the reaction rate.

The simulator

In this case, you will use a simulator to perform the kinetic experiments. This simulator is based on a mathematical model, and it is therefore "perfect", it does not result in any experimental errors. This is, of course, not how the outcome of an enzyme kinetic assay would work in real life, where such perfect data would not be obtainable.

References

  1. Lehninger, Albert L.; Nelson, David L.; Cox, Michael M. (2008). Principles of Biochemistry (5th ed.). New York, NY: W.H. Freeman and Company. ISBN 978-0-7167-7108-1.

Theory overview

Referred from:

Article
Enzyme Kinetics