ChBE

310 Homework 4

Assigned: 02/12/2015 - Due: 02/19/2015

Problem 1. An enzyme converts a substrate (S) into a product (P) through rapid and reversible formation
of an intermediate complex between the enzyme and substrate (with equilibrium dissociation constant
KM) and irreversible dissociation of the complex to form the product and release the enzyme (with rate
constant k2). Derive the rate expression of product formation in the presence of both a competitive
inhibitor (IC, with equilibrium dissociation constant KC) and an uncompetitive inhibitor (IU, with
equilibrium dissociation constant KU). Both inhibitors bind to the enzyme reversibly.




Problem 2. High-fructose corn syrup (HFSC) is a mixture of glucose and fructose commonly added to
foods and drinks for taste enhancement. Sucrose and fructose are monosaccharides obtained from the
hydrolysis of sucrose catalyzed by the enzyme sucrase.

The association and dissociation rate constants of sucrose-sucrase complex formation are 109 M-1min-1
and 1.2105min-1 respectively. The turnover number of sucrase is 105 min-1.
0.5 Kg of sucrose is added to a 5L solution containing 60 mg of sucrase (MW 180,000 g/mol):
a. How long will it take to degrade 90% of sucrose?
b. How long will it take to produce 0.25 Kg of fructose?



Problem 3. An enzyme converting substrate S into product P is immobilized onto calcium alginate beads
for continuous use in a bioreactor. The bioreactor containing 30 nM of immobilized enzyme is pretreated
in a buffer solution at 50C for 2 hours to eliminate contaminants from the immobilization reaction. The
temperature of the bioreactor is lowered to 37C prior to addition of the substrate.
The rate constant of enzyme denaturation at 50C is 0.05 min1 and the activation energy is 10 kcal/mol.
The enzyme is stable at 37C.
The Michaelis-Menten constant is 510-5 M and the turnover number is 104 min1.
a. How much substrate needs to be added to the bioreactor to achieve 99% substrate conversion in
10 hours?
b. How long will it take to achieve the same conversion if the bioreactor pretreatment is conducted
for 4 hours instead?
c.

How long will it take to achieve the same conversion if the bioreactor pretreatment is conducted
for 2 hours but at 60C?

ChBE 310 Homework 4

Assigned: 02/12/2015 - Due: 02/19/2015

Problem 4. Consider an enzymatic reaction in which an enzyme (E1; 1 M) is inhibited by its own
substrate at high substrate concentration. The equilibrium dissociation constant of enzyme-substrate
complex formation at the active site is 3.210-5 M and the equilibrium dissociation constant of enzyme-
substrate complex formation at the inhibitory site is 510-4 M. The turnover number of the enzyme is
103s-1.
a. Calculate the reaction rate if the substrate is 80 uM.
b. The enzyme E1 was engineered to generate an enzyme variant (E2) with decreased affinity at the
inhibitory site. As a result, the fraction of complex enzyme-substrate (in which the substrate is bound
at the inhibitory site of E2) is 1% of that obtained with E1. Calculate the reaction rate if the substrate is
80 uM.
c. Compare enzyme E1 and E2: plot of the reaction rate as a function of substrate concentration.
d. The enzyme E1 was also engineered to achieve partial substrate conversion at the low affinity binding
site (E3). As a result, substrate conversion occurs both at the high affinity binding site (with turnover
number 103s-1) and at the low affinity binding site (with turnover number 10 s-1). What is the reaction
rate if the substrate is 80 M?
e. Further engineering of enzyme E1 and E2 results in fully active enzymes E4 and E5, respectively, with
turnover number 103s-1 for both the high and low affinity sites. What are the reaction rates of E4 and E5
if the substrate is 80 M?
f. Compare enzyme E3, E4 and E5: plot of the reaction rate as a function of substrate concentration.



Problem 5. The antibiotic sulfanilamide is similar in structure to para-aminobenzoic acid (PABA), an
intermediate in the biosynthetic pathway for folic acid. Sulfanilamide can competitively inhibit the
enzyme that has PABA as it's normal substrate by competitively occupying the active site of the enzyme.
The equilibrium dissociation constant of PABA for the enzyme is 3 M.
You engineered a sulfanilamide variant (I1) with equilibrium dissociation constant ten times lower than
that of PABA.
a. Define the fractional measured enzyme effect. Calculate the IC50 of I1 if there is 7 M PABA in the
reaction.
b. You are investigating the enzyme mechanism and develop another inhibitor (I2) that binds to the
enzyme on a site other than the active site. I2 binds to the enzyme with the same affinity of I1 but
regardless of whether the enzyme is bound to PABA. You may assume that I2 presents the same
affinity for the unbound and PABA-bound enzyme. Define the fractional measured enzyme effect.
Calculate the IC50 of I2 if there is 7 M PABA in the reaction.