Why is enzyme kinetics important

Biochemical reactions are catalyzed by specific enzymes. Therefore, molecular and kinetic characterization of the enzymes that lead to the identification of the biochemical reactions constitute an important area of biochemistry. In kinetic characterization, kinetic constants of the enzyme are calculated, and the reaction mechanism can be deduced. The inhibitor kinetics may give information about the reaction mechanism; or it can be used in drug development [ 1 ].

Although the trivial names of some enzymes are more common as compared to their systematic names, the systematic IUPAC name, together with the EC number and the source it is purified from must be written in the introduction section. Of course, the reaction catalyzed by this enzyme and its importance for the organism is expected to be summarized, emphasizing the importance of the study presented.

The enzyme investigated could have been purified in the laboratory or purchased. If purified, the purification method must be summarized and cited; if purchased, the trademark must be given.

In either case, specific activity of the enzyme must be included. Then comes the summarizing of the activity measurement method and the definition of the enzyme activity unit.

Final concentration of each component in the assay medium, final volume and pH of the assay mixture and the working temperature must be specified. End point determination is not suitable for kinetic studies. Depending on the convenience of the measurement, either the rate of the increase in product [ 2 ] or the rate of the decrease in substrate concentration [ 3 ] is measured.

In some cases where neither measurement of the product nor the substrate is possible, coupled assays can be used [ 4 ]. Activity measurement is studied in the range where the enzyme obeys first order kinetics, in other words, in the range where the activity increases linearly by time. For this purpose, different concentrations of the substrate are prepared, usually in the range of 0. For each substrate concentration, time vs. Spectrophotometry is the preferred technique and most of the spectrophotometers have this graph plotting function, giving the linearity coefficient of the measurement as well.

Usually, measurement of this initial velocity where the concentration changes linearly with time can be completed by following the activity for about 1 min. The instrument used must be noted because a measurement limitation of the instrument is important. The initial velocities obtained are then used to plot the hyperbolic Michaelis-Menten graph V vs.

The latter is easier to show the kinetic constants [ 1 ], [ , 5 ]. However, several software is available for nonlinear curve-fitting, there is no need to take the reciprocals of V and S to find these constants. The software gives them directly from the Michaelis-Menten equation, together with the standard errors [ 6 ].

Lineweaver-Burk plot can be used in the laboratory for checking the results obtained while the experiment is running. In the manuscript, the graph may be required for a better presentation.

Measurement of the initial velocities of a hypothetical enzyme at four substrate concentrations S 1 , S 2 , S 3 , S 4. Increase of product concentration is plotted vs. For bisubstrate reactions, when one substrate is kept constant at various concentrations, the second substrate concentration is changed and vice versa [ 4 ]. Of course, increasing the number of measurements increases the accuracy.

Then the reaction mechanism can also be elucidated by nonlinear curve fitting. Assay conditions are determined by preliminary experiments. Usually working at optimum temperature and pH are preferred. But sometimes, if the enzyme turnover number is too high so that the first order phase cannot be detected or if substrate inhibition is high at optimum conditions, assays can be performed outside of the optimal conditions.

Attention must also be given to selecting the buffer. It must not be the substrate, product, inhibitor or activator of the enzyme. Suitable buffer and suitable buffer concentration may also be selected by preliminary experiments. As is known, kinetic constants obtained in inhibitor studies are called apparent Km Km app and apparent Vm Vm app. It was hypothesized that the optimal pH for the enzyme was pH 7 while the 1.

At the end of the experiment the results prove the hypothesis to be incorrect. These proteins are made up of monomers known as amino acids. The low bond dissociation energy formed by hydrogen bonding is what makes transferring of a proton more favorable within an enzyme. This allows the enzyme to achieve its characteristic features as a catalyst without undergoing any chemical reactions.

Lab Report -- Relationship on Enzyme activity and substrate concentration Research Question: Is the more concentrated the substrate of hydrogen peroxide is, the shorter the time taken for the paper disc to rise from the bottom of the beaker? Aim: The opposite of hull hypothesis Background Information: This experiment aimed to investigate on the relationship of the substrate concentration and enzyme activity. Enzymes are proteins produced by a cell that acts as catalysts to increase the rate of a specific chemical reaction without changing the reaction itself.

The movement of substrates into the cells compartments is also controlled by the energy charge. When sugar is added to the protein solution, the OH groups of sugars may also compete for hydrogen-bonding. The additive interacting more strongly with protein than with water will tend to stabilize the denatured states by the formation of protein additive complexes.

They will, therefore, have a denaturing effect. However, additives interacting more stronglywith water molecules than with protein will favour the stabilization of protein. Introduction: Enzymes are biological catalysts that increase the rate of a reaction without being chemically changed. Enzymes are globular proteins that contain an active site. A specific substrate binds to the active site of the enzyme chemically and structurally 4.

Enzymes also increase the rate of a reaction by decreasing the activation energy for that reaction which is the minimum energy required for the reaction to take place 3. Multiple factors affect the activity of an enzyme 1. Research Question How does the change in temperature affect the effectiveness of protease on breaking down egg whites? This textbook also outlines additional factors that contribute to the kinetics of reactions catalyzed by these proteins such as variability in isoforms pharmacogenomics and experimental factors including key concepts such as alterations of substrate concentrations due to binding.

Applications of these approaches in predicting kinetic parameters and alternative approaches for enzymes systems biology and transporters are also discussed.

The final section focuses on real-life examples case studies to try and exemplify the applications of enzyme kinetic principles.

This chapter provides a brief overview outlining some key concepts within each of the sections and the chapters within this textbook.