155:411 Introduction to Biochemical Engineering Fall 2012

Exam 1
October 10, 2012 from 5:00-8:00 p.m.

1. (15 points) Consider the peptide, biochemengisfun, whose sequence is represented as

follows:
NAla-Phe-Leu-Ile-Val-Arg-Ile-Gly-Leu-Ala-C

a. Find the net charge on biochemengisfun at

pH 7.
b. Imagine that this peptide can fold in such a
way as to bury exactly 3 successive amino
acid residues in its interior. Which sequence
of 3 amino acids from biochemengisfun
should be buried? Support your answer with a
calculation.

2. (20 points) The accompanying Scatchard plot

shows the binding of ethidium bromide (EtBr) to
DNA at two temperatures. The concentration of
EtBr is [A] and r is the amount of EtBr bound per
nucleotide of DNA. EtBr is a planar, aromatic
molecules that binds to DNA by intercalation
between adjacent base pairs.
a. What are the values of the dissociation
constant, Kd, and number of binding sites, m,
at 20 C?
b. At which temperature (0 C or 20 C) is the
affinity between EtBr and DNA greater?
c. At which temperature (0 C or 20 C) is the number of sites on DNA for EtBr binding
greater?

3. (25 points). The Lineweaver-Burk plot

beside shows the reciprocal of reaction
velocity as a function of (reciprocal)
substrate concentration in the absence
and presence of 10-4 M inhibitor (I).
a) What are the values of vmax and Km in
the absence of inhibitor?
b) What are the values of vmax and Km in
the presence of inhibitor?
c) What type of inhibition is this?
d) What is the dissociation constant of
this inhibitor (i.e., KI)?

4. (20 points) The G for the conversion

of fructose 1,6-bisphosphate (FBP) into
glyceraldehyde-3-phosphate (GAP) and
dihydroxyacetone phosphate (DHAP) is
+5.7 kcal/mol. This reaction is step four
of the glycolytic pathway and is
155:411 Introduction to Biochemical Engineering Fall 2012

catalyzed by aldolase. In the cell at 37 C, the mass action [DHAP]/[GAP] = 5.5

(a) What is the equilibrium concentration of [FBP] when [GAP] = 1.8 x 10-5 M, assuming
that the mass action between DHAP and GAP is maintained?
(b) What is the actual concentration of [FBP] when [GAP] = 1.8 x 10-5 M, given that the
observed G = -0.3 kcal/mol, assuming that the mass action between DHAP and
GAP is maintained?
(c) Since the standard state free energy for this step is positive, does it need to be
coupled to ATP hydrolysis to proceed spontaneously? Explain why or why not.

5. (20 points) An enzyme is surface-immobilized in microtiter wells. The wells are loaded
with different amounts of enzyme E0 and incubated with different substrate
concentrations S0, and reaction rates are assessed by accurately measuring accumulation
of product. The experiment is repeated for varying agitation speeds, with the following
results:
Average reaction rates in nmol/cm2/min
E0 = 0.05 nmol/cm2 E0 = 0.1 nmol/cm2
Agitation S0 (M) = 1 10 1 10
speed (rpm)
10 0.054 0.33 0.062 0.53
30 0.076 0.35 0.097 0.64
100 0.10 0.36 0.14 0.70
300 0.12 0.37 0.19 0.72
1000 0.13 0.37 0.23 0.74

(a) You decide that the two data points collected with E0 = 0.05 nmol/cm2 at the highest
agitation speed accurately reflect the intrinsic kinetics of the enzyme. State two
reasons why you are so sure.
(b) Use the data points specified in (a) to determine the kcat and Km for the immobilized
enzyme.
(c) Calculate the effectiveness factor for the sample at E0 = 0.1 nmol/cm2, S0 = 1 M, and
an agitation speed of 10 rpm.

Pledge: On my honor, I have neither received nor given any unauthorized assistance
on this examination.
________________________________________ (signature)
Potentially useful data:

Amino acid GEW (kcal/mol) GEW

(kcal/mol)
Gly -4.63 0
Ala -3.90 0.73
Val -2.94 1.69
Leu -2.21 2.42
Ile -1.69 2.94
Phe -1.98 2.65
Pro -2.06 2.57