Charles Larsen, Chemical Engineering
Introduction
During the 1960s, Dr. Thomas Brock studied life in the hot springs of Yellowstone National Park. There he found new strains of bacteria capable of living at temperatures above 75°C, which at the time represented the highest temperature at which life was known to exist. Dr. Brock’s discovery has sparked a world wide research effort to understand thermophilic bacteria, both their biochemistry and industrial applicability.
Under the direction of Dr. Gary Watt of Brigham Young University, I have studied the energy efficiency of selected metabolic reactions of thermophilic bacteria. The following report will outline the specific reactions I have studied, including a description of the methods I have used and plans for continuing the project.
MgATP Hydrolysis
The fundamental biochemical reaction by which an organism receives energy is the enzymatic hydrolysis of MgATP:
MgATP + Hp = MgADP + P + energy
I have proposed to measure the energy released by this reaction at temperatures near l00°C. This can be measured by combining a stream of MgATP with a high temperature enzyme stream in an isothermal flow calorimeter.
I have obtained a Chaperone enzyme from the thermophilic bacterium Thermococcus Litorallis. This protein will catalyze the hydrolysis reaction under appropriate conditions. I have measured the kinetics of the enzyme by conducting the experiment at 80°C and periodically sampling small portions of the reaction mixture for two hours. The amount of hydrolysis achieved is measured by injecting some of each sample into a High Performance Liquid Chromatograph (HPLC) and comparing the ratio of ATP to ADP present.
Under the conditions of my experiment, only 2% of the ATP will hydrolyze in two hours. I have two options now:
• increase the concentration of the enzyme
• try the experiment under anaerobic conditions
The supply of enzyme is limited, and it is difficult to get. I am currently designing a method for testing the enzyme kinetics in anaerobic high temperature conditions. The flow calorimeter is operated under anaerobic conditions and, if I can show that the enzyme is active anaerobically, the hydrolysis reaction will be straightforward when conducted by flow calorimetry. Beside the flow calorimeter a microcalorimeter that operates by a batch process can be used to measure the reaction energy. If the material requirement is too large to perform the experiment in the flow calorimeter, I will use the batch process. The enzyme kinetics and nucleotide concentration requirements will dictate which is most appropriate
Preliminary Reactions
Other reactions I have studied by calorimetry are binding of Mg2+ to ATP and ADP nucleotides and the protonation of these nucleotides. I have worked with Peming Wang, a graduate student under Dr. Reed Izatt, and with Dr. John Oscarson of the Chemical Engineering Department on the calorimetric measurement of these reactions. Two papers have been prepared for publication describing the results we have obtained.
Besides the calorimetric study of Mg2+/nucleotide binding, I have used a sephadex G-10 gel column to measure the equilibrium constant. From the equilibrium constant other important theimodynamic properties of the reaction, such as the Gibbs Free Energy, can be calculated. My data agree with the calorimetric data in that as temperature is increased the equilibrium constant of the binding reaction, both to ADP and ATP, increases. My values are lower than the calorimetric data and I believe the difference is due to ionic strength disparities between the different buffers. I am currently conducting the column experiment with the ionic strength adjusted to match that of the calorimeter solutions. The result might be available by Christmas, 1994.