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(a) Active site of an enzyme is the specific region of an enzyme where a substrate binds
and catalysis takes place or where chemical reaction occurs. The active site is more specific to
enzymes and refers to the site where the enzyme functions. The amino acids that form the active
site are located in distinct parts of the amino acid sequence of the enzyme. Therefore, the
primary structure of the enzyme should fold into its 3D structure, enabling the active site to come
together. Every enzyme contains one or more active sites .Substrate binding site along with the
catalytic site form the active site of the enzyme. The active site of an enzyme comprises a
substrate binding site that orient the substrate for catalysis. Binding site of an enzyme refers to a
region where inhibition reactions of enzyme and substrate occur. One or more substrate binding
sites can be found in an enzyme. Binding of the substrate causes the enzyme to adjust its shape
slightly , leading to a better induced fit. Allosteric site is the alternative binding site away from
the active site. Allosteric site often found in enzyme with 4o structure. The allosteric site allows
molecules to either activate or inhibit, or turn off, enzyme activity. These molecules bind the
allosteric site and change the confirmation, or shape, of the enzyme. Allosteric site comprises to
allosteric regulation which are allosteric activation and allosteric inhibition. The binding of an
activator stabilizes the active form of the enzyme. Conversely, the binding of an inhibitor
stabilizes the inactive form of the enzyme.

(b) i.) Competitive inhibitor is the inhibitor that competes with the normal substrate for binding
the active site of the enzyme. A competitive inhibitor is structurally similar to the normal
substrate and fits into the active site and combines with the enzyme. Competitive inhibitor
occupies the active site only temporarily and does not permanently damage the enzyme. This
kind of inhibition is reversible and can be overcome by increasing the concentration of substrate
so that as active sites become available, more substrate molecules than inhibitor molecules are
around to gain entry to active sites.

Non-competitive inhibitor has no structural similarity to the substrate and does not compete
directly with the substrate at the active site. It alters the structure of the enzyme in such a way
that the substrate may get attached to the active site but products are not formed. The reaction
goes on decreasing as more and more inhibitors contact the enzyme till saturation is reached. In
some case, it does not affect the ability of the substrate bind with the enzyme, but it makes it
impossible for catalysis to occur.

ii.) The term feedback inhibition refers to a situation in which the substances at the end of a
long series of reactions inhibits a reaction at the beginning of the series of reactions. The product
of one enzymatic reaction may control the activity of another enzyme , especially in a sequence
of enzymatic reactions. A different enzyme catalyzes each step, and the final product may inhibit
the activity of first enzyme. When the concentration of product is low , the sequence of reactions
proceeds rapidly. However, an increasing of concentration of product serves as a signal for the
first enzyme to slow down and eventually to stop functioning. Inhibition of the first enzyme stop
the entire reaction sequence.

Allosteric enzymes are a class of regulatory enzyme. Allosteric regulation is a type of

enzyme regulation by the reversible non-competitive inhibitions. Allosteric enzymes typically
have multiple active sites located on different protein subunits. When an allosteric inhibitor binds
to an enzyme, all active sites on the protein subunits are changed slightly so that they work less
well. There are also allosteric activators. Some allosteric activators bind to locations on an
enzyme other than the active site, causing an increase in the function of the active site. Also, in a
process called cooperativity, the substrate itself can serve as an allosteric activator, binding by a
substrate to one active site affects catalysis in different active site. This is considered allosteric
regulation because the substrate affects active sites far from its binding site.

iii.) Most enzymes have an optimal pH , at which the rate of reaction is the fastest. The
optimal pH for most human enzymes is between 6 and 8. Changes in pH may not only affect the
shape of an enzyme but it may also change the shape or charge properties of the substrate so that
either the substrate cannot bind to the active site or it cannot undergo catalysis. Extremely high
or low pH values generally result in complete loss of activity for most enzymes. Small changes
in pH above or below the optimum do not cause a permanent change to the enzyme, since the
bonds can be reformed. However, extreme changes in pH can cause enzymes to denature and
permanently lose their function.

2(a) i. Glycolysis begins when glucose undergoes phosphorylation, catalyzed by enzyme

hexokinase. Glucose receives phosphate group from ATP molecule and converted to glucose 6
phosphate. Once ATP is spent , it becomes ADP. The resulting phosphorylation of glucose
makes it more chemically reactive. Glucose-6-phosphate is then undergoes isomerization
become fructose-6-phosphate. Another ATP donates phosphate group to fructose-6-phosphate
and become fructose 1,6 phosphate. So far, this phase is known as energy investment phase that
2 ATP molecule are invested in these process. Phosphate group are now linked to carbon 1 and 6
and molecules is ready to be split. Fructose 1,6 phosphate is split into glyceraldehyde 3
phosphate (G3P) and dihydroxyacetone phosphate. The dihydoxyacetone phosphate is
enzymatically converted to G3P. Each G3P molecule is undergoes dehydrogenation with NAD+ .
The NAD+ gains electron (act as hydrogen acceptor) from G3P molecule and converted to
NADH. Then each G3P molecule reacts with inorganic phosphate in cytosol to form 1,3-
bisphosphoglycerate. One of the phosphate in each 1,3-bisphosphoglycerate react with ADP to
form ATP. The transfer of phosphate from phosphorylated intermediate to ATP is known as
substrate-level-phosphorylation. 3-phosphoglycerate is then undergoes isomerization into 2-
phosphoglycerate by enzymatically shifting the position of phosphate group in molecule. Next,
water molecule is removed , resulting in the formation of double bond and become the product,
phosphoenolpyruvate (PEP). A phosphate group is attached to the PEP by an unstable bond
(wavy line). Each of two PEP molecule is then undergo substrate-level-phosphorylation by
transferring its phosphate group to ADP in order to form ATP and form the product, 2 three-
carbon of pyruvate. Pyruvate formed in glycolysis is then enter in mitochondria, where they are
converted to acetyl coenzyme. Pyruvate undergoes oxidative decarboxylation by removing the
carboxyl group of molecule as carbon dioxide which diffuse out of the cell. This process is
catalyzed by enzyme pyruvate dehydrogenase. Then, the remaining two-carbon fragment is
oxidized and its electrons are transferred to NAD+ (hydrogen acceptor). The oxidized two-carbon
fragment, an acetyl group is attached to coenzyme A, forming acetyl coenzyme A. CoA has a
sulfur atom that forms a bond with acetyl group.

ii.) The unstable bond attaching acetyl group to coenzyme A breaks. Each two carbon acetyl
group combines with a four-carbon compound, oxaloacetate to form six-carbon compound citrate.
Coenzyme A now is free to combine with another acetyl group ad repeat the process. Citrate is
converted to its isomer, isocitrate through the process of removing and adding water molecules.
This isomerization is catalyzed by enzyme aconitase. Isocitrate undergoes decarboxylation and
dehydrogenation to yield 5-carbon compound, -ketoglutarate. The -ketoglutarate is then
undergoes decarboxylation and dehydrogenation to form succinyl coenzyme (4-carbon
compound) and catalyzed by enzyme -ketoglutarate dehydrogenase. Succinyl coenzyme A is
converted to succinate and substrate-level phosphorylation takes place . Breakdown of succinyl
coenzyme A coupled to phosphorylation of GDP to form GTP ( compound similar to ATP). GTP
transfer its phosphate to ADP to yield ATP. Succinate undergoes oxidation and transfers two of
its hydrogen to FAD, forming FADH2 , catalyzed by enzyme succinate dehydrogenase. The
compound formed is fumarate. Fumarate is converted to malate with addition of water. Malate
undergoes dehydrogenation , forming oxaloacetate . Two of its hydrogen are transferred to
NAD+, forming NADH. Oxaloacetate can reacts with another molecule of acetyl group, and a
new cycle beginning.

iii.) The reduced compound NADH and FADH enter the electron transport chain in the inner
mitochondrial membrane . The high-energy electrons of their hydrogen atom are passed down
from one acceptor to another. In the electron transport chain, each acceptor molecule alternately
reduced as it gains electron and oxidized as it transfer electron to another. The electrons are
passed along in a series of exergonic redox reaction , some energy is released in each step to
drive the synthesis of ATP molecules, which is an endergonic reaction. The process of ATP
coupled to the redox reaction in electro transport chain is known as oxidative phosphorylation.
The entire electron transport chain is divided into four large, protein complexes of acceptors.
Complex I (NADH-ubiquinone oxidoreductase) accepts electrons that are produced during
glycolysis,formation of acetyl CoA and the citric acid cycle. Complex II (succinate-ubiquinone
reductase) accepts electrons from FADH2 moleules that are formed during citric acid cycle. The
product of Complex l and Complex II , reduced ubiquinone become the substrate of Complex III
(ubiquinone-cytochrome oxidoreductase). Complex III accepts electron from reduced ubiquinone
and passed them to Complex IV (cytochrome c oxidase). Complex IV use the electrons that
accepted from cytochrome c to reduce molecular oxygen, forming water molecules in the process.
Oxygen molecules is the final electron acceptor in the electron transport chain.The electrons
simultaneously react with proton from the surrounding medium to form hydrogen. The chemical
reaction between hydrogen and oxygen produce water molecules.

Substrate-level- Processes of aerobic Reduced Of high energy Oxidative
phosphorylation (ATP ) respiration compound(NADH/FADH2) phosphorylation ( ATP)
2 ATP Glycolysis 2 NADH 4-6 ATP
(glucose pyruvate )

- Formation of acetyl CoA 2 NADH 6 ATP

(pyruvate acetyl CoA)

2 ATP Citric acid cycle 6 NADH 18 ATP

Total : 4 ATP Total : 32 - 34 ATP

Total number of ATP molecules produced during aerobic respiration :

Total ATP from substrate-level-phosphorylation + Total ATP from oxidative phosphorylation
= 4 + (32 - 34) ATP
= 36 - 38 ATP

(c)(i) The possible products of fermentation are alcohol or lactate. For lactate fermentation, glucose
undergoes glycolysis to form 2 pyruvate . During glycolysis, 2 NAD+ is reduced to 2 NADH and substrate-
level-phosphorylation takes place which phosphorylated intermediate transfer its phosphate to 2 ADP to
form 2 ATP. 2 NADH produced during glycolysis undergo dehydrogenation that transfer its hydrogen
atom to 2 pyruvate become 2 NAD+, yielding 2 lactates.
For alcohol fermentation , glucose undergoes glycolysis to form 2 pyruvate . During glycolysis, 2
NAD+ is reduced to 2 NADH and substrate-level-phosphorylation takes place which phosphorylated
intermediate transfer its phosphate to 2 ADP to form 2 ATP. 2 pyruvate is then undergo decarboxylation
by removing carboxyl group, releasing 2 carbon dioxide molecules and form acetaldehyde (2 carbon
compound). 2 NADH produced during glycolysis undergo dehydrogenation that transfer its hydrogen
atom to acetaldehyde become NAD+, yielding 2 ethyl ethanol.

(ii) Muscle cells undergo lactate fermentation due to the insufficient oxygen for aerobic respiration
occurs. The accumulation of lactate in muscle cells increase the acidity of the muscle cells. Lactic acid
built up in muscle cells lead to muscle cramps and soreness.
Faculty Of Applied Science
Diploma in Chemistry and Biology
AACB 1253 Bioenergetics

Assignment 1

Name : Liew Weng Yi

Student ID: 17WLD02258
Tutorial group : DCB I
Tutor name : Dr Lim Ah Kee