ChE513: Biochemical Engineering University of Santo Tomas Engr.

Achilles Espaldon

Submission Date: June 20, 2012

Assignment # 2 (for all sections)

1. The following kinetic data were obtained for an enzyme in the absence of inhibitor, and in the presence of two different
inhibitors and at 5 mM concentration (you are to determine which among the data below is with and without inhibition). Assume [Eo] is the same in each experiment. [1] [2] [3] [S]mM v(umol/mL.s) v(umol/mL.s) v(umol/mL.s) 1 12 4.3 5.5 2 20 8 9 4 29 14 13 5 35 21 16 12 40 26 18

a. Determine the vm and km for the enzyme. b. Determine the type of inhibition for the cases presented. c. Calculate the KI or K3.
d. Calculate the degree of inhibition.

2. Answer #13 in the file Enzyme kinetics examples and problems.

a. As an additional question, calculate the % or degree of inhibition for all types.

3. Liver alcohol dehydrogenase (ADH) is partially specific and will oxidize ethanol or other alcohols, including methanol.
Methanol (CH4O) oxidation yields formaldehyde, a toxic product, which can cause blindness. Mistaking it for a cheap wine which he usually prefers, your dog Pulgoso accidentally drank about 50 mL of Prestone De-Icer windshield washer fluid (a solution, 52% v/v of methanol). Knowing that methanol would be excreted in the urine eventually by Pulgosos kidneys if its oxidation could be blocked and inhibited, and realizing that, in terms of methanol oxidation by ADH, ethanol would act as a competitive inhibitor, you decided to offer Pulgoso some wine. How much of Pulgosos favorite Carlo Rossi wine (11.5% w/w ethanol) must he consume in order to lower the activity of his ADH on methanol to 5% of its normal value if the Km values of canine ADH for ethanol and methanol are 1 millimolar and 10 millimolar, respectively? (The KI for ethanol in its role as competitive inhibitor of methanol oxidation by ADH is the same as its Km). Express activity as reaction velocity. Both the methanol and ethanol will quickly distribute throughout Pulgosos body fluids, which amounts to about 15L. Assume the densities of both 52% methanol and the wine to be 0.87.
(my apologies to animal rights activists, if there are in the class)

4. Consider a substrate which is a weak acid, HA, with pKa = 4.0 and the ionized form A- =[S] is the one that can bind to
the enzyme. The enzyme, however, with pKa = 7 has an active site which contains a basic group that must be protonated in order to bind to the negatively charged substrate. a. Draw the mechanism of the enzymatic reaction b. Derive the equation for calculating the reaction velocity. c. At what pH will you set in order to maximize the concentration of both substrate and enzyme available for catalytic reaction? d. Plot the pH vs. %dissociation graph. e. Write the mass balance for the total enzyme concentration.

5. A new enzyme is being evaluated of its activity with respect to temperature. Experiment is executed by measuring its
activity and conducting it in the range of 5-800C at 5C0 interval. Initial enzyme concentration is 5x10-3M while activation energy of the reaction is 12 kcal/mole. During a kinetic study, constants where determined such as A = 25 1/s and the correlation obtained between the deactivation constant with temperature is given below;

k d = 2 x1012.e0.081.T
a. Plot the activity vs. temperature same time avoiding a denaturation or deactivation by operating below the Topt.

b. Determine the optimum temperature necessary for running an enzymatic reaction in high activity and at the

"I pledge on my honour that as the group representative, I have honestly accomplished this homework and assignment only with the help of my groupmates and that sharing our solutions, answers and ideas to other groups violates the principle of independent thinking and working which constitutes our core integrity as students of ChE513 Biochemical Engineering. I believe that doing my best effort with the right mindset and attitude will fulfill my highest ideals as a student of this university."

____________________________________ Signature of group representative

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