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.
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,
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.
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.
- 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.