Practice Exams

BIOC 307/407 Exam 1

Name__________________________________
2a. Name the structural motif represented in the figure and specify: left/right handed, or parallel/antiparallel,
as appropriate for this motif. Explain the reasons for your answer.

2b. Draw an arrow to indicate the position of one of the peptide bonds in the structure.
2c. Indicate the alpha carbons of the amino acids on either side of this bond. Label with .
2d. For this peptide bond, individually circle each atom that is part of a plane.

3.

) Michaelis-Menten kinetics, short answer.

3a. Show the simple reaction scheme that applies to the enzymatic conversion of S to P in the
Michaelis-Menten treatment of enzyme kinetics. Include the appropriate rate constants.

3b. What is the ES complex usually called?

3c. Express the initial velocity rate of the reaction as a function of [ES]. Dont do anything
complicated, as no equations containing more than 4 terms will receive credit!
3d. How does Vmax depend, if at all, on the amount of enzyme in the reaction? Do not use the
Michaelis Menten equation even if you memorized it!
3e. How does kcat depend, if at all, on the amount of enzyme in the reaction? Explain your answer.
Do not use the Michaelis Menten equation even if you memorized it!
3f. Apply the principle of conservation of mass to the substrate concentration. Indicate what
simplification can be made if [S] >> [ET], as is usually the case.

Practice Exams

BIOC 307/407 Exam 1

Name__________________________________

(8 points) Inhibition of enzymatic reactions.

vo

[S]
There are four curves on the graph. One shows V0 in the absence of inhibiton. The others are for
increasing [Inhibitor].
Label curve for reaction without inhibitor as .
Label curves as

in order of increasing inhibitor concentration

Which curve if any represents the greatest apparent value of KM. Explain.

Which curve if any represents the greatest apparent value of Vmax. Explain.
.
What type of inhibition is this? Explain.
5

ATCase (asp transcarbamoylase) catalyzes the reaction: asp + carbamoyl

phosphate carbamoylaspartate + phosphate

5a Explain feedback inhibition, using ATCase as an example. Correctly use the words committed,
chain and allosteric in your explanation.

5b. Succinate,
is an inhibitor of ATCase. Indicate what type of
inhibitor, and why you think that is the case.

Practice Exams

BIOC 307/407 Exam 1

Name__________________________________
6

The folded structure of a globular protein in aqueous solution.

6a. Indicate one interaction, or thermodynamic driving force, that stabilizes the folded structure.
Explain how this stabilizes the structure of folded proteins.

6b. Indicate one interaction, or thermodynamic driving force, that destabilizes the folded structure.
Explain how this is destabilizing.

6c. What kind of amino acid side chains are typically found in the interior of a protein active in
aqueous solution?

Practice Exams

BIOC 307/407 Exam 1

ID #__________________________________
No text on the back of any page will be graded
All answers need to be 15 words or less: only first 15 words will be graded
1. (9 points) Structures of amino acids:
1a. Draw the structures of the following amino acids (2 points each):
L-Ala

D-Thr

L-Asn

Starting at the COO and going around clockwise, you get CO R N for L and CO N R for D. Some
people drew the H of the alpha carbon in a vertical bond. As the H then points backward in the
Fischer projection, things were more complicated: You had to now go around anticlockwise.
1b. Indicate the names of the following amino acids (no D/L needed) (1 point each).

Lys

Leu

Met

2. (6 points). Examine the protein structures below. For each of the two structures, indicate 1) the most prominent
secondary structure and 2) the featured higher order structure

Practice Exams

BIOC 307/407 Exam 1

ID #__________________________________
1)2) a two (or more) domain protein1) 2)

3. (12 points) Michaelis-Menten kinetics, short answer.

Consider the simple reaction:

S-> P
k1

k2

Catalysis is in 2 steps:

E+P
k-1

k-2

The Michaelis-Menten equation: vo = Vmax [S]

KM = (k-1 + k2)/k1

[S] + KM
3a. (2 points) What is the ES complex usually called?
Michaelis complex, partial credit for enzyme substrate complex. NO credit for transition state!
3b (2 points) Under which circumstances can the back reaction of the second step be ignored?
Right after mixing enzyme and substrate, when [P] can be ignored. No credit for special cases with
particular rate constants.
3c (2 points) What is the practical meaning of KM?
When [S] equals KM, vo = Vmax. KM = Vmax is an incorrect expression. This can be seen as the
units on both sides of the equation (M and M/s, respectively) are not the same.
3d (1 point) Write a simple equation for Vmax (Hint: it does not contain [S]).
Vmax = k2[ET]
3e (1 point) In words, indicate why enzymatic reactions have a Vmax.
At high [S], the enzyme gets saturated with S. Then further addition of S will not lead to further
increase in vo. This plateau is Vmax.
3e (4 points) kcat
) Give the definition of kcat
kcat = Vmax/[ET]
) What is the common name for kcat
The turnover number
) Why is the above a useful expression?
The expression above (3e ) defines a quantity (kcat) that is [ET] independent. This can be used for
comparison of different enzymes. Vmax is dependent on [ET] and not useful in this regard. For credit
it was sufficient to mention that kcat was useful for comparison to other enzymes. Mention of the use
of kcat in kcat/KM (the catalytic efficiency; needed to show this equation) also was good for credit.
) What is the practical meaning of kcat?
The number of catalytic reactions carried out per enzyme per second, when [S] is saturating.
4. (6 points) TS analogs are potent competitive inhibitors of enzymatic reactions
4a Without invoking any specific examples, explain what transition state analogs are.

Practice Exams

BIOC 307/407 Exam 1

ID #__________________________________
TS analogs are stable compounds that emulate the structure of the substrate(s) in the active site of the
enzyme for the TS. For full credit you needed to point out this similarity. Partial credit for
mentioning similarity to the substrate(s)
4b Why is it that TS analogs are competitive inhibitors?
TS analogs are no substrate for the enzyme, but due to their similarity to the substrate(s), the TS
analogs bind to the active site and as such are competitive inhibitors. For full credit, the active site (or
catalytic site) needed to be mentioned.
4c Why is it that they are potent inhibitors?
TS analogs are potent inhibitors as they bind tightly to the active site, taking advantage of the special
interactions of the enzyme particular to the TS. It is not correct to state that the TS analog binds
irreversibly to the enzyme however. Partial credit for just mentioning tight binding to the enzyme
without reference to the TS. No credit for essentially repeating the answer for 4b.
5

(6 points) RNAase A

The RNAase A catalyzed reaction is carried out in two separate steps. See a schematic of RNase As
active site in the figure below. The red arrows only

5a How is the reaction initiated? Draw an arrow to show this. A

5b What happens next? Draw another arrow to show. B
5c Clearly indicate in the figure which bond in the RNA is broken to split the RNA into two parts.
C: The P O bond at the tip of lightning bolt.
5d Circle the part of the RNA which next dissociates from the RNase A enzyme D
For full credit, the O coming off of the P O bond needed to be included.
5e What next happens to part of the space previously occupied by the RNA that has dissociated?
Practice Exams

BIOC 307/407 Exam 1

ID #__________________________________
A molecule of H2O partially fills this space.
5f How is the second step of the reaction initiated?
His 119 gets the H+ back by taking it form the H2O that just came in.
6. ( 8 points) Protein folding/unfolding
6a. Indicate two ways other than addition of urea, by which one can get a folded protein to unfold.
1) Heat; 2) extremes of pH; 3) Guanidinium HCl; 4) SDS
6b When returning the unfolded protein to conditions that favor folding, the re-folding step goes
through an intermediate.
( ) What is this intermediate called?
The molten globule
( ) What are two salient features of the intermediates structure?
1) It has some secondary structure
2) It has exposed hydrophobic groups
( ) What undesirable property does folding intermediate have?
Aggregation with other similarly incompletely folded proteins
( ) How does the cell deal with this problem?
Chaperones provide unfolded or partially folded proteins an environment for folding in isolation,
so they wont aggregate with other proteins. The answer chaperones by itself, without indicating
that they are folding cages, received partial credit.
6c What is an amyloid fiber?
A fibrous structure consisting of aggregated, misfolded or partially folded proteins.
6d Name a disease that is caused by protein misfolding.
I had prion diseases of various kinds, and Altzheimers disease in mind, but cystic fibrosis and
sickle cell anemia also got credit.

Practice Exams

BIOC 307/407 Exam 1

Practice Exams

BIOC 307/407 Exam 1

Practice Exams

BIOC 307/407 Exam 1

Practice Exams

BIOC 307/407 Exam 1

Practice Exams

BIOC 307/407 Exam 1

BIOCHEMISTRY 307/407 Exam 1

Your 7-digit ID:_______________________________________
100 points total: 8 exam questions = 85 points

1 home work assignment = 15 points

Use only the space provided. Do not write on the back of any page. It will not be graded.
Some useful equations:

vo =

Vmax [S]
K M + [S]

Go = -RT lnKeq

KM =

k1 + k 2
k1

1. (10 points). Amino acids.

a. Identify the following 3 amino acids (Full name or 3 letter abbreviation)

Trp

Arg

Met

b. Draw D-asparagine

2. (10 points). Michaelis-Menten:

Refer to the scheme at right:
a. If k-1 >> k2 for an enzyme which of the above steps is rate limiting for the formation of P?
The second step: ES E+P
The answer that it has to be E + S ES is not correct. The reason that most of the reactions go back to
ES is that k2 is small is that ES E+P is very slow, and rate limiting. The answer ES is also not correct
because by itself ES is not a step.
b. For the same enzyme, where is the transition state for the rate limiting step in the scheme?
Between ES and E + P, or more correctly, between ES and EP. The answer ES E+P with no further
indications, is not clear and received only one point
c. For another enzyme with k2 >> k-1, which of the above steps is rate limiting for the formation of P?
The first step: E + S ES
d.) Why is kcat called the turnover number?
Because it indicates how fast the enzyme can turn over the substrate. Correctly formulated: the
maximal number of substrate that each enzyme (or more precisely each catalytic site of the enzyme) can
convert to product per second.

Practice Exams

BIOC 307/407 Exam 1

BIOCHEMISTRY 307/407 Exam 1

e) Why is kcat/KM called the catalytic efficiency?
It indicates how efficiently the enzyme catalyzes the substrate to
product when [S] << KM, which is the case for the living cell. The
higher the value of kcat/KM, the more reactions will be catalyzed
per second under these conditions.
3. (10 pts) RNAase.
Offer a plausible explanation for the data shown in the figure on the
right for the catalytic activity of an RNAase. Over the range shown,
the pH does not affect the tertiary structure of either the RNAse or the
RNA.
The shape of the curve arises from the involvement of the
above two histidines in the catalysis.
At very low pH each of the histidines is positively charged
due to a bound H+. The rate is close to 0. Upon addition of
some base, first one histidine is titrated to give up its H+.
From the fact that the rate drastically increases, we know it
must be the histidine that has to be neutral for RNase A to function. It now can bind an H+.
Upon further titration the rate decreases steeply.
In this pH range, the H+ is next removed from the histidine
that needs to be positively charged. Once both histidines are
neutral, the rate of catalysis is negligible again: there is no
longer an H+ to be donated by the second histidine!
Some people got confused and took the curve to mean that the pH changes during the reaction. This is not the
case! Several reactions were set up to check what would happen to the reaction at different pH.
4. (15 points) Protein folding (this question concerns proteins in aqueous solutions, not membrane proteins).
a. An important intermediate in protein folding is:
The molten globule
b. Mention two characteristic features of this intermediate.
1) Secondary structure has formed
2) Many nonpolar groups are still exposed.
c. What problems are posed by the presence of (partially) unfolded proteins in a cell?
The main problem is that nonpolar groups are exposed to the aqueous solution. Hydrophobic
interactions between unfolded proteins will result in irreversible formation of inactive protein
aggregates.
d. Mention one condition that would cause such unfolded proteins or partially folded intermediates to
accumulate.
My intended answer included: Heat; high and low pH; denaturants like urea.
Many people read condition as an ailment (e.g. heart condition) instead. For full credit, the name
of the disease needed to be mentioned, as well as the fact that it involved misfolded proteins.
e. What proteins assist in overcoming these problems? How are (partially) unfolded proteins distinguished
from fully folded proteins?

Practice Exams

BIOC 307/407 Exam 1

BIOCHEMISTRY 307/407 Exam 1

Here the context is important, as the header indicates these problems, i.e. exposure of nonpolar
groups. Thus chaperones would be involved, which, with their folding cages, allow unfolded proteins to
fold in isolation so that the problem of aggregation is avoided. The cavity of GroEL/S chaperones has a
nonpolar rim which can interact with the exposed nonpolar region of the unfolded protein, so that it will
be delivered to the interior of the folding cage.
5. (8 pts). Draw an S-S bridge between two cysteines that are in the same polypeptide chain (not at either end
and not right next to each other). Show all atoms of both cysteines. Show the rest of the main chain as a line
and label the N and C termini of the chain.
For full credit, you needed to satisfy all the above criteria, and have it correct see figure below. There
are no charged groups in the main chain except for 1 at each end!

6.Secondary structure (8 points).

a. Secondary structure (alpha helix and beta structure) is stabilized by hydrogen bonding between groups of
the main chain. Sketch such a hydrogen bond and indicate the donor and the acceptor.
N-H...O=C
Donor
Acceptor
b. Sketch a polypeptide chain containing two parallel stretches of beta structure (shown as broad arrows) and
the rest of the main chain connecting them (shown as a line). Do not show the H-bonding.
The connector has to be from the bottom of
one arrow to the top of the other in order to
maintain the correct N - C direction in each arrow.
Some people connected the two bottom or the two
top parts of the structure. This results in an
impossible structure as N C would be
divergent for the two section of beta structure of
the same chain: or .

Practice Exams

BIOC 307/407 Exam 1

BIOCHEMISTRY 307/407 Exam 1

7. The Michaelis Menten equation (12 pts)
a. Use the graph to estimate the values of the following parameters
(with units):
By eye, Vmax = 600 M/s and KM = ([S] when v0 = Vmax) = 5M.
By fitting the curve, Vmax = 650 M/s and KM = 6 M.
b. Indicate how the data points shown in the figure were obtained.
This is not about how Vmax and KM were determined. It is about
the date displayed in the figure. For each point on the graph, a
solution had been prepared containing S at a concentration
shown on the x-axis. Enzyme (at the same [ET] for all [S] ) was then added and the production of P was
followed as a function of time immediately after the start of the reaction. The initial velocity was
measured from the slope of the curve ( [P] on the y axis vs time on the x-axis) through the origen (i.e. for
t = 0). This value of vo was then plotted against the [S] to get the curve shown in the figure.
c) Does catalysis affect the concentrations of S and P at equilibrium? Explain.
No. The difference between the standard free energies of P and S is the only parameter that sets the
equilibrium constant: Go = -RT lnKeq, where Keq = [Peq]/[Seq] = k1/k-1. Catalysis reduces the activation
free energy of the TS (G). It does not in any way affect Go, or Keq, or the distribution of S and P at
equilibrium. You needed to mention the equilibrium constant not changing, or just speeding up the rate
of getting to equilibrium for full credit.
Several people mentioned that at equilibrium the forward and backward rates were the same, so that
the equilibrium could not be changed. This is not true, because you could have different concentration
of P and S at equilibrium and still have the backwards and forwards rates be the same due to the fact
that the action of the enzyme had affected the forward or backward rate constants or both.
d) Does catalysis accelerate just the forward reaction or both the forward and the reverse reactions (i.e. is only
formation of P accelerated or both formation of P and S)? Explain
Catalysis takes place in both directions as the activation free energy of the TS (G) is reduced by the
enzyme, affecting catalysis in both directions. You could also look at Keq, it is the ratio of the forward
and reverse rate constants: Keq = [Peq]/[Seq] = k1/k-1. As Keq is not affected due to catalysis, the ratio of
rate constants does not change either. This means that if the forward rate constant (k1) goes up by, for
example million fold, the reverse rate constant (k-1) has to also increase by the same factor in order for
Keq to stay constant. Full credit for any similar reasoning.
e) Use the expression for KM (top page 1) for an enzyme with a single substrate, to derive the units for KM.

KM =

k1 + k 2
The units for k-1 and k2 are s-1 (first order) and for k2 M-1s-1(second order). The units for
k1

KM can be determined as (s-1 + s-1)/s-1M-1. The s-1 all cancel and 2/M-1 is left, which, in units, is M.

8. (12 points) Aspartate transcarbamoylase (ATCase) catalyses the following reaction:

aspartate + carbamoyl phosphate carbamoylaspartate + H2PO4-

Practice Exams

BIOC 307/407 Exam 1

BIOCHEMISTRY 307/407 Exam 1

a. Where on the ATCase do the substrates
(aspartate and carbamoyl phosphate) bind?
To the catalytic sites on the C (catalytic)
subunits.
b. Where on the ATCase do ATP and CTP bind?

ATCase: Allosteric effectors and cooperativity

Purified catalytic
trimer

To the regulatory subunits, which clearly

constitute an allosteric site (other than the
active site).
c. Purified trimers of the catalytic subunit exhibit
Michaelis-Menten kinetics, but the intact ATCase
does not. Explain.
That the C3 follow M&M is not surprising, as it
does not have attached regulatory subunits. So why
does ATCase not follow M&M, even in the absence
of any ATP and/or CTP (see figure above)? ATCase
has 2 states, T (inactive) and R (active). In solution
ATCase by itself is mostly T. Binding of substrates
effects a concerted transition from T to R: all C
subunits change state together. This makes the
transition cooperative. The higher the
concentration of substrates, the more R will be in
solution. Because this cooperative T to R transition
is dependent on [S], when increasing [S] you have to
first deal with the T state, and then with the R state.
The vo obtained for ATCase in the T state is much
lower than that obtained for the R state. This is the
reason that ATCase does not follow M&M (see slide
# 71 of lectures 7-11, shown on the left).
d Explain how ATP or CTP act on ATCase to regulate its activity.
ATP binds at the r subunits, and causes conformational changes in both the r and C subunits which pull
ATCase towards the R state. It is an activator of ATCase.
CTP also binds at the r subunits, but it it causes other conformational changes in the r and C subunits
which now pull ACTase towards the T state. It is an inhibitor of ATCase

Practice Exams

BIOC 307/407 Exam 1