A cost-effective enzyme kinetics and inhibition model for biochemistry education and research

Abstract

Enzyme kinetics and inhibition studies are crucial in biochemistry education and research. Conventional methods often require expensive equipment and reagents, potentially limiting their accessibility in limited resource settings. Our approach sought to develop a cost-effective experimental design for studying enzyme kinetics and inhibition. Lactase was chosen as a protein model and its activity was investigated by measuring glucose production from lactose hydrolysis. In the study, commercially available lactase pills were used as an enzyme source, while milk was used as a substrate. Instead of scientific equipment, glucometers were used to measure lactase activity. Enzyme kinetics were evaluated using Michaelis-Menten and Lineweaver-Burk plots. In the study, the effects of temperature, pH, and inhibitors were also investigated. The results of our study aligned with established enzyme kinetics theories and previous studies. Lactase showed temperature and pH-dependent activity, with decreased activity observed at both low and high extremes. Results also showed that galactose acts as a competitive inhibitor of lactase. The approach presented here offers a cost-effective procedure for studying enzyme kinetics and inhibition. It can act as a valuable tool for educational purposes and for preliminary research in settings with limited resources.

Keywords: Michaelis–Menten; biochemistry education; enzyme kinetics; glucometer; inhibition; lactase; milk; pH and temperature dependence.

Figure 1.

A. The structure of lactase (β-galactosidase). It is a tetramer and each subunit can bind to a lactose molecule and catalyze its hydrolysis. B. One monomer of lactase showing galactose bound to the active site. C. Reaction catalyzed by lactase. Conversion of lactose into galactose and glucose. Note that the galactose also acts as an inhibitor. D. Beta galactosidase active site showing galactose and the magnesium ion. PDB ID 1JYV.

Figure 2.

Enzyme kinetic lab in a grocery bag. A. Milk containing lactose for the enzyme-substrate B. Measuring strips for the glucometer. C. Galactose for lactase inhibition D. The lactase pills to serve as the enzyme source E. Glucometer to serve as the instrument to measure enzyme activity. F. Cost breakdown for a lab of 8 students working in pairs.

Figure 3.

Steps involved in the cost-effective kinetics experiment using lactase. A. serial dilution of the milk solution in the PBS buffer or water to offer six different lactose concentrations. 2. Grinding the lactase tablet using a motor and pestle. 3. Adding the lactase powder into the milk and stirring. 4. Take an initial reading and another one every 2 minutes until the reaction is over (10 minutes total). Repeat until activity in all six beakers with different concentrations are measured.

Figure 4.

Michaelis-Menten kinetics of lactase. A. Michaelis-Menten curve showing the progression of different enzyme rates at different substrate concentrations. B. Lineweaver-Burk curve showing a Vmax of 8.57mM/min on the Km of 54.25mM. C. Time course of all of the five experiments at different substrate concentrations.

Figure 5.

Effect of temperature on enzymatic activity. A. Lactase activity at 4 degrees Celsius. B Lineweaver-Burk plot of lactose activity at 4°C vs. room temperature. It shows that the Km varies but the Vmax stays the same. C. Michaelis-Menten curve of lactase activity at 37° Celsius versus room temperature. d Lineweaver-Burk plot of lactase activity at 37° C.

Figure 6.. pH

effect on enzymatic activity. A. Enzymatic activity of lactase in milk over time at different pH readings. pH 6.7 pH 8 and pH 10. B. Comparison of enzyme activity at pH 6.7, 8, and 10.

Figure 7.

Inhibition of lactase by the galactose. A. Schematic representation of lactase structure and active site with galactose bound. B. 3D Structure of Beta galactosidase bound to galactose. PDB ID 30B8. C Graphics done using Protein Imager.[38]Michaelis-Menten curve of lactase with and without inhibitor. D. Lineweaver-Burk of the galactose inhibition of lactase.