Determining the Cause of Peak Invertase Activity Delay using Isothermal Titration Calorimetry

Abstract

Invertase catalyzes the hydrolysis of sucrose producing glucose and fructose. However, its mechanism of action is not well understood as other studies have shown that there is an initial delay before the enzyme reaches peak activity. This indicates that there may be another step for the active form of invertase to be activated. To better understand this mechanism, isothermal titration calorimetry (ITC) was used to measure the enzymatic activity of invertase. In this study, varying concentrations of invertase between 0.0125 to 0.125mg/mL were used to react with 1M and 2M sucrose solutions. Results show an exponential relationship between varying concentrations of invertase and the time to reach peak height. Increasing 0.0125mg/mL of invertase by tenfold, decreases the time required for the enzyme to reach peak activity by 85%. This suggests at increased invertase concentrations there is more active invertase, which may be because invertase forms a type of multimer for full activity. The effect of temperature was also explored, 1.2M sucrose was titrated into 0.016mg/mL invertase at temperatures from 25 to 55 °C. By comparing 25 and 45°C, we observed that the time required to reach peak activity decreased by 62%. However, at 55°C, the time increased by 24%. Also contributing to the delay is the viscosity of sucrose. Concentrations of sucrose ranging from 0.5 to 2M were titrated into 0.0125mg/mL of invertase. The time for the enzyme to reach peak activity increases by 23% when increasing sucrose concentration from 0.5M to 2M. However, this increase in time was also observed in the control (0.5 to 2M sucrose concentration titrated into 0.1M sodium acetate buffer). Therefore, the viscosity of sucrose does not completely account for the delay. In conclusion, the delay in peak maximal activity is in part due to increased sucrose concentration, probably due to inefficient mixing. Additionally, the delay in maximal activity is decreased in the presence of increasing invertase concentration indicating that there are inter‐invertase interactions that are required for maximal invertase activity.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.