Biochemistry 100 Level Study Questions and hints/answers. Enzymes.

1. Enzyme Catalysis
a. What is a catalyst?
Some material (molecule/ion/surface/mixture) that increases the rate of a
reaction and is unchanged at the end of the reaction.

b. What is altered in an enzyme-catalysed reaction compared to an uncatalysed reaction

and how does this change increase the reaction rate?
The activation energy is decreased so the reactant/substrate molecule(s) do
not need as much energy for the reaction to occur (proceed to the transition
state) so the reaction proceeds (products made per time) at an increased rate.

c. Draw a diagram representing this concept.

d. What is the transition state?

The short-lived unstable (highest free energy) state on the reaction pathway
(more similar to the product than the substrate is.

e. What is not altered by catalysis?

DG for the reaction, the position of equilibrium, the structure/properties of
the catalyst.

2. Enzyme Active Sites and Specificity

a. Define the term ‘enzyme active site’. In general terms, what does the active site consist of?
A part of the tertiary (can be quaternary sometimes) structure of an enzyme
that has complementarity in both shape (structure) and chemistry (e.g.
polarity/charge/hydrophobicity) to the substrate(s) which allows the
substrate(s) to bind i.e. closely interact (orientation & proximity)

b. In general terms, describe ways in which enzymes catalyse reactions so that they occur so
much faster than they would without the involvement of the enzyme.
Enzymes bind substrate(s) in the correct orientation and position for
productive collisions (reaction) to occur more frequently. The active site
provides amino acid functional groups and sometimes cofactors that interact
with the substrate to promote transition state stabilization.
 Some enzymes form covalently bonded E-S intermediates that are more
reactive than the substrate (as in chymotrypsin).
Enzymes can stretch/bend bonds (strain effects) because of interactions with
functional groups on essential active site residues with those of the substrate
– this would help the rearrangement of electrons within bonds of the
substrate(s).
The enzymes can provide a different chemical environment (to aqueous
solution) inside the active site (hydrophobic or charged) to promote the
reaction.

c. What is meant by specificity of an enzyme and what types of specificity are there?
An enzyme usually catalyses a single chemical reaction, or a set of closely
related chemical reactions. An enzyme’s specificity is the type of substrate
that will interact with its active site and result in a reaction and the reaction
it catalyses.
Broad specificity: a number of molecules of the same general type
(e.g. Chymotrypsin catalyses the hydrolysis of peptide bonds C-terminal to
phenylalanine ANY protein containing these amino acids, but the bond-type
and location is specific)
Absolute specificity: Only a single compound can bind
(e.g. Glucokinase will only bind glucose)
Stereospecificity: Only the correct optical isomer of the compound will bind
(not covered in 100 level but is a possible answer). This can be common,
because of the chirality of carbon compounds and the 3D shapes of active sites
and substrates.

d. Explain why an enzyme might be stereospecific (extension question;not required/covered in

lectures)
The stereospecific substrate has to fit into the active site for the reaction
to occur. The enzyme active site will have complementary shape to a
particular stereoisomer of the substrate molecule, while another
stereoisomer of the same molecule is unlikely to fit correctly (just like you
can’t easily fit a right hand into a glove made to fit a left hand).

e. What is the specificity of chymotrypsin? What type of enzyme is chymotrypsin and where
is it found? Chymotrypsin is a digestive protease that hydrolyses a peptide bond
C-terminal to a phenylalanine in proteins. It is produced in the pancreas in an
inactive zymogen form and transported to the small intestine where it is
activated and digests proteins in food. Chymotrypsin is a member of the serine
protease (proteinase) family.

3. The Active Site and Mechanism of Chymotrypsin

 a. What are the three essential catalytic amino acid residues (catalytic triad) of
chymotrypsin? (residue numbers don’t need to be memorised).
i) Serine 195

ii) Histidine 57

iii) Aspartate 102

iv) Which of these residues actually interacts with the substrate molecules?
Serine 195 with the protein and Histidine 57 with a water molecule

v) What part of the chymotrypsin active site determines its specificity?

The hydrophobic pocket – the hydrophobic phenylalanine side chain fits in
this site

b. Use reference material (and/or the figure below) to write the hydrolysis reaction (single line
format) producing two products from one initial polypeptide. Consider the protein substrate
as two ‘R groups’ either side of the peptide bond e.g. R-CONH-R’

R-CONH-R´ + Enz ® RCOO- + NH3+R´ + Enz

protein enzyme N-terminal peptide product C-terminal peptide product

c. Study the diagram below showing aspects of the chymotrypsin enzymatic

mechanism. The substrate has been simplified (from Biochemistry Berg et al,
wikibooks). You can refer to the figure in the notes/Appling for additional help if
needed. This question is largely over to you.
 i) For step 1 label the catalytic triad residues on the figure (Q3a). Describe how the
environment of the catalytic triad modifies their amino acid properties enabling
chymotrypsin to act as a catalyst.

You should be able to identify the catalytic triad serine (OH containing side chain),
histidine (cyclic N-containing side chain) and aspartate (carboxylic acid -containing
sidechain).
The histidine is able to accept a proton from the serine as is it a basic amino acid and
because the now positively charged protonated histidine is stabilized by ionic
interaction with the negatively charged aspartate. The deprotonated Ser is now
activated to act as a nucleophile (electron rich group) that can now form a transient
bond with the substrate.

ii) The chymotrypsin mechanism of substrate specificity is not illustrated in the figure on
the previous page. Modify the figure to show the mechanism for chymotrypsin substrate
specificity and explain how this specificity is achieved.

Draw a hydrophobic pocket on the RHS of the active site that can accommodate R1
which is the phenylalanine side chain

iii) Referring to the figure describe how chymotrypsin is able to promote transition state
stabilisation.

Highlight how the ‘oxyanion hole’ (see the figure), which comprises chymotrypsin
groups with positive electrostatic potential, (details not shown in the figure above)
forms stabilising electrostatic interactions with the negatively charged oxyanion
intermediate. The Ser by covalently attaching to the substrate and His by
orienting/positioning the hydrolytic water molecule can be considered here also.

iv) Predict (not covered in lectures but you might be able to come up with justified
hypothesis/es) how the activity of chymotrypsin could be sensitive to pH.

The catalytic mechanism requires Ser 195 to donate a proton, that is accepted by His 57
so that the Ser can react with the substrate. However, if at low pH (high [H+]) the His 57
is already protonated then it will not be able to accept a proton from the Ser so the
enzyme will be inactivated.

Now a real assessment question from a previous year (and you should find it easy after completing
everything above)

Name the three active site amino acids comprising the chymotrypsin ‘catalytic triad. Briefly
explain how each is involved in the catalytic mechanism of the enzyme. Which part of the active
site structure determines the specificity of chymotrypsin for only certain peptide bonds? (5 marks)

Give it a go yourself. The residue numbers are not necessary if you can’t remember them, but
you need to know which amino acids are needed, and the involvement of each. If you are
unsure, read what it says in the textbook to explain the diagram and in the text. Then try again.
Note the mark allocation – you don’t need to give a complete breakdown of the whole
mechanism as well as what you are specifically asked above for 5 marks = 5 minutes. But you
should write at least one sentence about each – and perhaps one extra for the most important
residue(s)

4. Enzyme Kinetics
a. Sketch a graph showing the dependence of reaction rate on substrate concentration for a
Michaelis-Menten enzyme (sometimes called a ‘classical’enzyme).

b. Show how you would determine KM and Vmax from this graph.
Vmax by extending line back from the maximal flat (saturated) region of the
hyperbolic curve until it meets the Y-axis.
For KM work out Vmax/2 then from this point on the Y-axis move right until
this value of Y meets the curve, then KM is the X-value that results in
Y=1/2Vmax in units of the X-axis.

c. What are the units of KM and Vmax? How would you work this out in a test if you had
forgotten to learn it? (think about the definitions of each as well)
KM has units of concentration (mole L-1 or µmole L-1) Vmax has units of rate
(e.g. µmole L-1 min-1)
They are read off the axes, use the same units as the axis they are shown on.

d. What is the significance of KM? What does a low KM value mean?

KM is a measure of the affinity of the enzyme for its substrate. A low KM
means that it binds strongly at very low concentrations of substrate.
Note that if an enzyme has several substrates the KM will probably be different for each.

e. If a reaction rate is equal to Vmax, what is in excess in the reaction mixture?

Substrate.

f. What changes in reaction conditions would you expect to change the measured Vmax?
Changes in enzyme concentration, temperature & pH. Also uncompetitive or
 mixed inhibitors. Possibly other conditions e.g. salt strength.

g. What is enzyme activity?

The rate of the reaction. The molar amount of substrate used or product
produced per unit time per amount of enzyme.

5. Enzyme Inhibition
The following graph shows the effects of a drug acting as an inhibitor on the activity of a viral enzyme.

a. Add some suitable example units to each axis.

e.g. Y-axis µm/min, X-axis mM

b. Which of Vmax, KM, or both, or neither, are affected by the inhibitor? Indicate on the graph
the approximate values for Vmax and KM for each reaction set (each curve).
Vmax is the same for the uninhibited and inhibited reactions. KM has increased in the
presence of the inhibitor

c. Is the inhibition shown above reversible or irreversible? What can you conclude about the
type of bonds that are formed between the inhibitor and the enzyme?
The inhibition is reversible, typically no or very little activity is seen when irreversible
inhibitors are present.

d. What class of (ir)reversible inhibition is consistent with the graph and the answers 5b & 5c
above?
Competitive inhibition (reversible) as Vmax is the same and KM has increased in the presence
of the inhibitor.

e. What does this class of inhibition mean with respect to the binding site of the inhibitor on the
enzyme?
The competitive inhibitor competes with the substrate for binding to the active site.

f. What can be concluded regarding the structural and/or chemical properties of the inhibitor
compared to the substrate?
 There is usually similarity in structure and chemical properties of the competitive inhibitor
and the substrate if they can both bind to the enzyme active site.

6.
a. State a different mechanism (class) of reversible inhibition for a Michaelis-Menten enzyme from that
answered in 5 above.
Uncompetitive or Mixed inhibition

b. How you would distinguish this second type of inhibition from that in Q5 with reference to Vmax?
Vmax decreases for both uncompetitive and mixed inhibition

c. What is the relationship between the substrate and inhibitor binding sites on the enzyme for this (Q6)
class of inhibition?
For either uncompetitive or mixed the inhibitor binds at a different site to the substrate on the
enzyme (i.e. not at the active site)

d. What is the relationship between the substrate and inhibitor structures and chemical properties for this
class of inhibition?
For either uncompetitive or mixed there is not necessarily any similarity to the substrate (there are
some special cases!)

Controlling (Regulating) Enzyme activity

7. State two ways in which the activity of an enzyme (that is already present in the cell) can be
regulated.
Allosteric control
Covalent Modification

8. Allosteric enzymes

a. What type of curve of rate vs substrate concentration is obtained for an allosteric enzyme?
Sigmoidal curve

b. What can you conclude regarding the quaternary structure of an enzyme if it displays
allosteric kinetics?
That it has quaternary structure

c. Sketch a graph (labelled axes) to show the effect of allosteric effectors on an allosteric
enzyme.
 d. Using this graph as a guide, write a description of how enzyme activity changes with
[S] and the effect of the different types of effectors.
Allosteric effectors change the affinity of the enzyme for its substrate
resulting in a change of reaction rate. A l l o s t e r i c inhibitors (negative
allosteric effectors) decrease substrate binding to the enzyme so a higher
concentration of substrate is needed to achieve half of Vmax, whereas
activators increase the amount of binding so the enzyme works faster at
lower concentrations of substrate.

e. Binding of allosteric effectors is reversible. What determines when an effector will

bind?
The concentration of the effector in the cell (or cell compartment) and its
affinity for the enzyme.

f. Why are allosteric effectors important in cells.

Because they have a nearly instantaneous effect on enzyme activity, allowing
for fast control. As binding is reversible, they allow pathways to be controlled
by controlling enzyme activity rapidly and responsively.

g. (Summary of above) Explain what is meant by allosteric regulation, allosteric effectors,

and effector binding sites. How does this regulation work?
One for you to do yourself

Regulation of Enzyme Activity by Covalent Modification

9. Explain, (you may use a diagram(s)) how the following two regulatory mechanisms involving covalent
modification control enzyme activity:

a. Phosphorylation

A protein kinase enzyme covalently attaches a

phosphate group from ATP (forming ADP) to a
specific amino acid (Ser, Thr, Tyr) side chain
on the enzyme. This charge change alters the
active site structure/properties and activates
or inactivates (depending on the enzyme) the enzyme. The enzyme can be restored to
its original state by removal of the phosphate by a group of enzymes called
phosphatases. The phosphate is released as the free ion (Pi), ATP is not reformed

b. Zymogen activation
In order to prevent enzyme activity in the wrong tissue or circumstances some
enzyme are synthesised as inactive zymogens. The zymogen is activated to the mature
enzyme when the enzyme is in the correct location e.g. when digestive enzymes are
secreted out of the pancreas into the small intestine, or e.g. activation of blood
clotting enzymes only once they are at the location of the wound that needs clotting
and not elsewhere in the circulatory system. The zymogen is activated by proteolytic
hydrolysis of certain peptide bonds in the enzyme main chain that cause a change in
the enzyme active site in some way that activates the enzyme. The enzyme cannot be
inactivated by the reverse of this process, the peptide bond cleavage is irreversible.