Calorimetric Measurement of Pseudo-First-Order Enzyme Kinetics
An ITC experiment is performed to measure dQ/dt after each substrate addition, using conditions to maintain steady state. For this method, it is recommended that KM be greater than 10 µM. If KM if lower than 10 µM, the continuous assay is suggested.
The recommended enzyme concentration range in the ITC cell is 25 pM to 1 mM. In general, the higher the enzyme-substrate affinity, the lower the enzyme concentration. The recommended substrate concentration range in the ITC syringe is 10 µM to 100 mM, it needs to be above KM and in excess of enzyme concentration in cell. Suggested injection parameters are 2 µL per injection and 15 injections.
The suggested interval between injections is three minutes to establish a new thermal power baseline before the next injection. This change in thermal power baseline is due to heat produced (or absorbed) from the enzymatic reaction; the ITC cell requires different power levels to maintain thermal equilibrium. As a general guideline, no more than 5% of the substrate should be converted before the next injection.
After completion of the experiment, Origin 7.0 software for ITC automatically performs data analysis. dQ/dt is determined by measuring the difference between original baseline and new baseline after each injection (Figure 2a). [S]t and [E] are also calculated after each injection. DHapp is determined in a separate ITC experiment. Using Equation 4, Rt is determined for each [S]t and data fit to Equation 2 to solve for kcat and KM.
Figure 2a shows ITC data for the titration of PP1-g phosphatase with PNPP. The power generated (dQ/dt) at each substrate concentration is proportional to the rate of reaction. Data from Figure 2a are used to determine the reaction rate at different substrate concentrations, and these are plotted in Figure 2b. As a comparison, data generated from spectrophotometric assays are also shown. Kinetic parameters obtained by the two assay methods are in excellent agreement. Since many substrates of protein phosphatases cannot be monitored with a spectrophotometer, ITC can be useful to study enzyme kinetics and mechanisms as well as in the design of therapeutic agents.
The steady-state ITC method has been used to determine the enzyme kinetics parameters of several enzymes, including H. pylori urease, E. coli GroEL chaperonin, heparinase I, yeast hexokinase, HIV protease, pyruvate carboxylase, and dinucleoside polyphosphate hydrolase. Values determined by ITC agree with enzyme parameters determined by other methods.