BIOCHEMISTRY MIDTERM REVIEW

(The following answers are only suggestions. You are encouraged not to memorize them word for word, but rather getting the main ideas and
then express them with your own words.)

1. Enzyme is widely used in many fields as food processing, environment, agriculture, medicine, …student
chooses one and discuss the application of enzyme.
(The following examples are only suggestions. You are encouraged to find your own examples, or build more into an example that you find
interesting.)

Examples of enzyme applications in:

a. Food processing: Fish sauce is a traditional savory in Vietnam and the craft of making it is an income source
for many households in coastal provinces. Traditionally, the process of making fish sauce takes a very long
time (12-24 months) because it relies on the naturally-occurring proteases in fish guts. By using additional
enzymes, such as alcalase and flavourzyme, the production time is cut down to half, the total nitrogen content
is higher, and the production cost is lower.
b. Environment: The production of dairies and textiles discharges a lot of dangerous waste into the environment.
This is a serious health hazard to people working in the factories as well as people living near the factories.
Enzymes, such as catalase, can play a role in degrading toxic wastes, including formaldehyde, formic acid and
alcohols, in a speedy and cost-effective way.
c. Agriculture: Enzymes are used widely in animal feeds production in order to improve the nutritional values.
Hydrolases are the most commonly used for this purpose, including amylase, proteinase, cellulase and
hemicellulase. These enzymes can break down large polymeric molecules in the raw materials into smaller
molecules that can be easily absorbed by even newborn or young animals, such as calves, chicks, puppies, etc.
d. Medicine: Enzyme diagnostics is a new branch of enzymology and medicine, which uses enzymes to
determine the concentration of components in blood, serum, urine, etc., such as glucose and cholesterol.
Additionally, determination of enzyme activity in biological materials is a useful indicator for damages to
vital organs.

2. Coenzyme is helper molecule and it takes an important role in enzyme catalyzed reaction. Discuss about it.
Coenzyme works as an assistant to enzymes. Such helper molecules can be in the form of a small organic molecules
or a metal ion. When a coenzyme is attached tightly to an enzyme by covalent bonds, it is called a prosthetic group.
Coenzymes facilitate the enzymatic reactions in several ways:
- Metal ions can bind different parts of the substrate to warp it into a more stable conformation. They can also accept
electrons from the substrate and stabilize the transition state. Moreover, the positive charges of the metal ions help
stabilize the transition state by shielding negative charges of the substrates.
- Organic molecules, often derived from vitamins, contribute to and determine the type of reaction by accepting or
donating electrons (redox reactions) or other chemical groups. With the help of these coenzymes, the enzymes
themselves are not altered after the reaction even if there is a net transfer to or from the substrates to form the
products.
The important role of coenzymes explains the need for consuming micronutrients from food, such as minerals and
vitamins. A lack of coenzymes lead to a corruption of enzymes function, as most enzymes require coenzymes, and
they cannot perform their catalytic activity without their coenzymes. This manifests into various diseases, especially
metabolic ones.

3. Active site occupies a small part of enzyme protein but it is the place where substrate comes and combines
with. Base your understanding, clarify the structure and property of active site.
Almost all enzymes have a protein nature, a large polymeric molecule with one or several polypeptide chains.
However, where the substrates bind and reaction takes place is only a small part of an enzyme’s total volume. As a
protein is ultimately folded into a 3D form, the active site is also three dimensional, often in the form of clefts or
crevices on the surface of the enzyme. The amino acids present in the active site are actually far away in their order in
the polypeptide chain, but brought close together after protein folding. This explains why enzymes lose their catalytic
activity when denatured, or when their active site is damaged. It also explains why isoenzymes, though arising from
different gene, can catalyze the same reaction with the same substrate, as long as their active sites after protein folding
are similar. The arrangement of residues in the active site also contributes to its specificity.
When a substrate comes into an active site, it is bound by multiple weak attractions with the amino acid residues,
cofactors and coenzymes, and even water molecules present in the active site.
4. Write down six categories of enzyme. Discuss and focus on the first three groups.
Six categories of enzymes: (remember to write them in the correct order)
1. Oxidoreductases
2. Transferases
3. Hydrolases
4. Lyases
5. Isomerases
6. Ligases
a. Oxidoreductases: catalyze the oxidation-reduction (redox) reactions. Their subclasses include
dehydrogenases, oxidases, oxygenases, reductases, peroxidases, and hydroxylases (For more details, see slides).
These enzymes contain coenzymes such as NAD+, NADP+, FAD, FMN, etc. that can accept or donate
electrons.
Dehydrogenases: Examples are alcohol dehydrogenase (turning alcohol into aldehyde with the help of
NAD+, seen in physiological degradation of alcohol) and glutamate dehydrogenase (turning glutamate into
ketoglutarate, seen in ammonia fixing microbes and mammals’ amino acid metabolism).
Reductase: Transfer of electrons to oxygen so that it can combine with H+ to form water molecules.
Oxigenase: Reactions that involve oxygen in a functional group such as OH, COOH, etc.
Peroxidase: Reactions that involve hydroxy peroxide (H2O2). The common coenzyme for this
subclass is heme. Catalase is a special example of this subclass, as its substrates are H2O2 only.
b. Transferases: Transfer of groups, such as amino, carboxyl, carbonyl, methyl, phosphoryl, acyl, etc. Their
names often include -trans-. The subclasses of these enzymes are very different in nature, depending on the
functional group that they transfer.
c. Hydrolases: Cleavage of bonds by adding water (hydrolysis). Some subclasses are esterases, phosphatases,
and peptidases.
(For other categories, see slides)

5. Can you recommend the enzyme source?

Enzyme production can come from 3 main sources: animals, plants and microbes.
Enzyme Sources Applications
Animal Sources
proteases bovine and porcine digestive enzymes, anti-inflammatory agents,
pancreas health food additives
pepsin porcine stomach body fortifying agents
aldolases liver and muscle fructose digestion
catalase liver food industry
chymotrypsin pancreas leather industry
lipase pancreas food industry
ancrod snake venom anti-coagulant
urokinase urine thrombolytic agent
alkaline phosphatase calf intestine/kidney diagnostic (indicator in ELISA)
Plant Sources
bromelain pinapple anti-inflammatory agents, meat tenderizer
papain papaya anti-inflammatory agents
urease jack bean determination of blood urea nitrogen
amylase barley brewing industry
peroxidase horseradish diagnostic
Microbe Sources
 cellulase Trichoderma break down of cellulose in detergent and paper
industry
amylase Bacillus subtilis break down of starch in industrial raw materials
protease Bacillus degradation of protein content in detergent and
food industry
alcohol dehydrogenase yeast diagnostic
L-asparaginase E. coli leukemia treatment

6. What do you know about enzyme kinetics? Discuss Vmax and KM, equation of Michaelis Menten and
Lineweaver Burk. Advantages and disadvantages of each?
Enzyme kinetics is the study of the rates of enzymatic reactions and how they can be affected by other conditions.
When an enzyme is added to a substrate, the reaction that follows occurs in three stages with distinct kinetics:
Phase Concentration of ES Rate of product formation
Pre-steady state Rapid burst of ES Initially slow as ES starts being formed, then
complexes formed speeds up
Steady state ES concentration remains Constant rate of formation, faster than pre-
constant as it is being steady state. This is the rate expressed by
formed as quickly as it Michaelis-Menten kinetics.
breaks down
Post-steady state Substrate runs out so ES Slow down as there are fewer ES complexes
concentration declines
Michaelis-Menten kinetics is a model of enzyme kinetics that explains the dependence of reaction rate on the
concentration of enzyme and its substrate. There are two important terms in the Michaelis-Menten equation:
V max [S ]
v=
K M +[S]
- Vmax: the maximum reaction rate, when all active sites are saturated with substrate. It is dependent on total enzyme
concentration.
- KM (Michaelis constant) – the substrate concentration at which the reaction rate is half of the Vmax. KM is an
important term because it measures the affinity an enzyme has for its substrate. The lower the KM value, the more
efficient the enzyme is at carrying out its function at a lower substrate concentration.
However, Vmax and KM do not depend on each other. Knowing KM does not predict Vmax and vice versa.
Plotting a graph of substrate concentration against the rate of the reaction gives a rectangular hyperbolic curve as
below. It can be seen how the reaction rate initially increases rapidly in an almost linear fashion as substrate
concentration increases (1st order kinetics). The rate increase then plateaus, and becomes less sensitive to substrate
concentration (0 order kinetics).

Michealis-Menten equation represents exactly the behaviour of reaction rate as substrate concentration increase.
However, it poses difficulties in experimental design, as it is hard to determine KM and Vmax from it.
Lineweaver-Burk equation is the reciprocal of Michaelis-Menten equation. It is more useful in determining KM and
Vmax thanks to its linear nature. By finding the x- and y-intercepts, we can easily work out the Vmax and KM values.
The Lineweaver-Burk equation is as followed.

1
=
( )
KM 1
+
1
v V max [ S ] V max

However, experimental design needs to be carried out with care, since the Lineweaver-Burk plot poses two
disadvantages: At large [S] (small 1/[S]), data points crowds together, making the resulting curve unreliable. At small
[S] (large 1/[S]), small errors, both human and systematic, can have large effect on the resulting curve.
7. Role and functions of PPP.
The pentose phosphate pathway (PPP) is a metabolic pathway parallel to glycolysis. Its main roles are to generate
NADPH and 5-carbon sugars, a precursor for nucleic acid synthesis called ribose 5-phosphate. PPP involves oxidative
and non-oxidative phases. The first phase is the oxidation of glucose 6-phosphate and the synthesis of NADPH. The
second phase is the non-oxidative rearrangement of carbon skeletons and 5C sugars synthesis. Therefore, PPP serves
as a bridge between carbohydrate metabolism and nucleic acid metabolism.
See lecture slides and book, try to memorize the reaction scheme for each step. You should be able to remember the
names of the intermediates in these scheme, and enzymes catalysing each reaction, and recall in which reaction
NADPHs are produced.
8. What do you know about glycolysis, focus on structure of intermediates, enzymes, NADHs and ATPs.
Glycolysis is the first step of glucose metabolism, where a molecule of glucose is turned into two molecules of
pyruvate. It includes 10 steps, divided into two stages: energy investment (steps 1-5) and energy payoff (steps 6-10).
Glycolysis occurs in cell cytoplasm.
See lecture slides and book, try to memorize the reaction scheme for each step. You should be able to draw the
chemical structure of the intermediates in these scheme, remember the names of intermediates and enzymes catalyzing
each reaction, and recall in which reaction ATPs or NADHs are consumed or produced.
9. What do you know about C.A.C (Krebs cycle), focus on structure of intermediates, enzymes, CO2, NADHs,
FADH2 and ATPs.
Citric acid cycle (CAC) occurs in mitochondrial matrix. After glycolysis, pyruvate is transported through the
mitochondrial membranes into the matrix and undergoes oxidation into acetyl-coA, which then joins the CAC.
CAC is a closed cycle, with the last reaction regenerates the molecule used in the first reaction (oxaloacetate). It
includes 8 steps.
See lecture slides and book, try to memorize the whole scheme of CAC, including the number of carbon molecules in
each intermediate, where CO2 is cleaved away, and where NADH, FADH2 and GTP are produced. You should be
able to draw the chemical structure (or at least a string of balls representing how many carbons there are) of the
intermediates, know the names of the intermediates and enzymes catalyzing each step.
10. What is metabolism and why do you need to study it? Base on your best understanding of metabolism,
summarize the key notes related to its basic roles and functions in living organisms
Metabolism is the sum of all the chemical changes that convert nutrients into energy and the complex finished
products of cells. Its basic roles include:
- To obtain energy from fuel molecules or sunlight
- To convert nutrients into precursors of cellular components
- To assemble precursors utilizing energy into cellular components
(Think of examples for each role)

9. Discuss and exemplify the differences between anabolism and catabolism, where possible.
Create a table with the following key words for catabolism and anabolism.
Catabolism: degradative pathways, energy-yielding, decomposition of large complex molecules into small molecules,
oxidative, converging
Anabolism: biosynthetic pathways, energy-requiring, construction of large complex molecules from small molecules,
reductive, diverging
(Try to elaborate on each key word)

12. Focus and discuss the energy generating pathways of carbohydrate metabolism.
Include 3 pathways: glycolysis, CAC and oxidative phosphorylation
As the question requires, focus on how much energy is liberated by each pathway, and the total amount of energy
yielded.
Glycolysis: After investing 2 ATPs in the first stage (steps 1 and 3), the second stage generates 4 ATPs (steps 7 and
10). So, glycolysis yields a net 2 ATPs for each glucose.
CAC: As the primary energy generated from CAC is contained in the reduced coenzymes, there are only 2 GTPs
(equivalent to 2 ATPs) produced in step 5 of the cycle that can be counted as liberated energy.
Oxidative phosphorylation: This is the pathway that generates the most usable energy for the cell. For a total 10
NADHs and 2 FADH2s from glycolysis and CAC, oxidative phosphorylation can generate up to 34 ATPs (3 ATPs for
each NADH, 2 ATP for each FADH2 – simplified calculation), or 28 ATPs (2.5 ATPs for each NADH, 1.5 ATPs for
each FADH2 – calculated based on numbers of protons pumped).
In total, one molecule of glucose can generate up to 38 ATPs or 32 ATPs after complete oxidation through three
pathways.

13. What is Biochemistry and its covering scopes as well as its applications in life sciences?
Answer based on your understanding.