Some comments on enzyme kinetics studies

Introduction

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].

Correct results require a precise study and sufficient knowledge. Here, I try to point out some aspects of the enzyme kinetics studies I noted when I had been assigned as a reviewer for several journals.

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.

Experimental

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. Enzyme activity definition sentence may have the following pattern: “One unit of enzyme activity is defined as the amount of enzyme required to generate (or consume) 1 (micromole) of substance X per unit time (usually 1 min) under standard assay conditions.” This statement obviates to define the standard assay conditions. Final concentration of each component in the assay medium, final volume and pH of the assay mixture and the working temperature must be specified. Specific activity is in terms U/mg protein or U/g wet weight of tissue. Although most of these points are usually listed in the “information for authors”, I find it helpful to reiterate them here.

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 0.3–2.0 Km. For each substrate concentration, time vs. activity is plotted and the initial velocity of the reaction where activity increases linearly with time is measured (Figure 1). 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. S) or linear Lineweaver-Burk plot (1/V vs. 1/S). 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.

Figure 1:

Measurement of the initial velocities of a hypothetical enzyme at four substrate concentrations (S₁, S₂, S₃, S₄). Increase of product concentration is plotted vs. time and the initial rates are calculated when product concentration [P] increases linearly with time. These initial velocities then (V₁, V₂, V₃, V₄) can be used to plot hyperbolic Michaelis-Menten graph (V vs. S) or linear Lineweaver-Burk graph (1/V vs. 1/S) or can be used in nonlinear regression analysis.

For bisubstrate reactions, when one substrate is kept constant at various concentrations, the second substrate concentration is changed and vice versa [4]. It is a good practice to prepare a matrix of at least 4 × 4 of substrates and make each measurement at least twice. 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). Same is the case when crude preparations or partially purified enzymes are investigated, since the test samples may contain inhibitors, activators or some other enzymes with similar activities. Under these conditions kinetic constants obtained are somehow different as compared to those of the pure enzyme, and therefore they are called Km_app and Vm_app.

Use of a pure enzyme is especially important for inhibition studies. First the enzyme activity is measured at increasing concentrations of the inhibitor. Next the initial velocity is measured at selected inhibitor concentrations. For reversible inhibitors, the inhibition must not be time dependent [2], [4], [7]. The type of inhibition and the inhibitor constants are deduced by various techniques [1], [, 6].

The last but not the least, the units must be checked for correctness.

Conclusion

Kinetic constants change with the purity of the enzyme. The presence of inhibitors or activators as impurities, presence of enzymes which produce or consume the substrates or products may change the results. Then, the more purified the enzyme, the more reliable is the kinetic study. I did not include the mathematical equations and the definitions of the kinetic constants here. They can be found in classical textbooks [1], [, 5]. I hope these comments would be helpful to the researchers in their future work.