BIOCHEMICAL ENGINEERING ENZYME KINETICS

1.) You carried out an enzymatic reaction in 5 L CSTR. The inlet substrate concentration was 100 mmol/L and the flow rate was set 1 L/hr. After a steady state was reached, the outlet substrate concentration was 10 mmol/L. a. What is the reaction rate in the reactor? b. You measured steady state outlet substrate concentration as a function of the inlet flow rate and found the following results. Estimate the Michaelis kinetic parameters by using the best plotting technique for the equal weight of all data points.

2.) A substrate is converted to a product by the catalytic action of an enzyme. Assume that the Michaelis Menten kinetic parameters for this enzyme reaction are: KM = 0.03 mol/L rmax = 13 mol/L min a. What should be the size of a steady state CSTR to convert 95 percent of incoming substrate (CSo = 10 mol/L) with a flow rate of 10 L/hr? b. What should be the size of the reactor if you employ a plug flow reactor instead of the CSTR in part (a)? 3.) A substrate is decomposed in the presence of an enzyme according to the Michaelis Menten equation with the following kinetic parameters: KM = 10 g/L rmax = 7 g/L min If we operate two one liter CSTRs in series at steady state, what will be the concentration of the substrate leaving the second reactor? The flow rate is 0.5 L/min. The inlet substrate concentration is 50 g/L and the enzyme concentration in the two reactors is maintained at the same value all of the time. Is the two reactor system more efficient than one reactor whose volume is equal to the sum of the two reactors?

4.) Eadie (1942) measured the initial reaction rate of hydrolysis of acetylcholine (substrate) by dog serum (source of enzyme) and obtained the following data:

Evaluate the Michaelis Menten kinetic parameters by employing (a) the Langmuir plot, (b) the Lineweaver Burk plot, (c) the Eadie Hofstee plot, and (d) non linear regression procedure. 5.) In order to measure the enzyme activity and the initial rate of reaction, 5 mL of cellobiose (100 micromol/mL) and 44 mL of buffer solution were placed in a stirred vessel. The reaction was initiated by adding 1 mL of enzyme (beta glucosidase) solution which contained 0.1 mg of protein per mL. At 1, 5, 10, 15, and 30 minutes, 0.1 mL of sample was removed from the reaction mixture and its glucose content was measured. The results were as follows:

a. What is the activity of the beta glucosidase in units/mL of enzyme solution and in units/mg protein? A unit is defined as the enzyme activity which can produce 1 micromol of product per minute. b. What is the initial rate of reaction? 6.) Eadie (1942) measured the initial reaction rate of hydrolysis of acetylcholine (substrate) by dog serum (source of enzyme) in the absence and presence of prostigmine (inhibitor), 1.5 x 10 -7 mol/L and obtained the following data:

a. Is prostigmine competitive or noncompetitive inhibitor? b. Evaluate the Michaelis Menten kinetic parameters in the presence of inhibitor by employing the Langmuir plot 7.) Invertase hydrolyzes cane sugar into glucose and fructose. The following table shows the amount of sugar inverted in the first 10 minutes of reaction for various initial substrate concentrations. The amount of invertase was set constant.

A Lineweaver Burk plot of the preceding data did not result in a straight line when the substrate concentration was high. To take into account the substrate inhibition effect, the following reaction mechanism was suggested:

a. Derive the rate equation using the Michaelis Menten approach. b. Determine the three kinetic parameters of the equation derived in part (a) using the preceding experimental data.

8.) The initial rate of reaction for the enzymatic cleavage of deoxyguanosine triphosphate was measured as a function of initial substrate concentration as follows:

a. Calculate the Michaelis Menten constants of the above reaction. b. When the inhibitor was added, the initial reaction rate was decreased as follows:

Is this competitive inhibition or noncompetitive inhibition? Justify your answer by showing the effect of the inhibitor graphically. 9.) The effect of an inhibitor on an enzyme reaction was studied by measuring the initial rates at three different initial inhibitor concentrations. The obtained Michaelis Menten kinetic parameters are as follows:

a. Write the kinetic model for this enzyme reaction. b. Derive the rate equation. State your assumptions for any simplification of the rate equation. c. Estimate the value of inhibition kinetic parameter.

10.) An enzyme (cathepsin) hydrolyzes L glutamyl L tyrosine to carbobenzoxy L glutamic acid and L tyrosine. It has been found that the glutamic acid formed in the hydrolysis, inhibits (competitively) the progress of the reaction by forming a complex with cathepsin. The course of the reaction is followed by adding tyrosine decarboxylase which evolves CO2

Calculate (a) the value of Michaelis Menten constants of the enzyme, KS, and (b) the dissociation constant of enzyme inhibitor complex.