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Briefly describe the mode of action of cellulase.

Cellulase lowers the activation energy for the cleavage of glycosidic bonds between B-glucose.

According to the Induced Fit Hypothesis, when cellulose binds to cellulase at its active site, it induces a
conformational change in cellulase such that cellulose can fit more precisely into its active site and forms an
enzyme-substrate complex which

maintains precise orientation of substrate molecules as enzyme molecules hold substrate molecules in the
correct orientation, to increase likelihood of effective collisions between substrate molecules

induces stress in bonds of substrate to increase likelihood of bond breakage within substrate

increases substrate reactivity by changing charge on substrate, since catalytic residues are very close to
part of substrate, to facilitate bond creation and breakage for reaction between substrate molecules.

Explain the mode of action of enzymes in terms of specificity and activation energy. (10)
Enzymes has a highly specific catalytic action due to the 3-D configuration of its active site.

The active site is made up of contact residues and catalytic residues.

The catalytic residues are responsible for the ability of the enzyme to catalyse a particular metabolic reaction
and they act on the bonds in the substrate.

The contact residues are responsible for the specificity of the enzyme and form a shape that is
complementary to the shape of the substrate.

According to the Lock and Key Hypothesis, substrates with a shape complementary to that of the active site
of an enzyme fits the enzyme like a key to a lock.

Binding of the substrates to active site of the enzyme forms a enzyme-substrate complex that bring the
substrates in close proximity & optimum orientation for bond formation.

In this way, the enzyme lowers the activation energy required for the reaction to occur, increasing the
number of substrate molecules with sufficient energy for reaction to occur and hence the rate of reaction.

Once the products are formed, the products no longer fit into the active site and leave the active site free to
be reused since they are not chemically changed at the end of the reaction.

Describe the effect of compounds x and y on the rate of nucleotide extension. (5)
X is a co-factor/ inorganic ion required for moulding the enzyme into its correct conformation. No proper
functioning of the enzyme in the absence of x.

Y is a competitive inhibitor. When Y is present, the rate of enzyme-catalysed reaction is lowered. However,
the rate of reaction is restored at high substrate concentrations. Inhibitor has the same shape as substrate,
and competes with substrate for the limited enzyme active sites.

Describe and explain the effect of mercury ions on the rate of reaction catalysed by
lactate dehydrogenase. (4)
In the presence of 10g/ml of mercury, the maximum rate (Vmax) of reaction decreased from 0.048 arbitrary
units to 0.033 arbitrary units.

Mercury ions act as noncompetitive inhibitor, and has a different structure from substrate, binding
irreversibly to the allosteric site of the enzyme and not the active site of the enzyme.

Mercury ions form strong covalent bonds to an amino acid R group on the enzyme, causing a
conformational change to the enzyme such that it alters the shape of its active site.

Hence, a portion of the enzymes is rendered non-functional, decreasing the rate of formation of
enzyme-substrate complex and hence, decreasing the rate of reaction.

Increasing substrate concentration will not allow the maximum rate of reaction to be attained.

Explain how HIV protease inhibitors work in treating HIV infection.

HIV protease cleaves HIV polyprotein into several functional proteins such as enzymes Integrase, Reverse
transcriptase and HIV protease which are responsible for formation of mature viruses.

HIV protease inhibitor is a competitive inhibitor that is complementary to and binds to active site of the HIV
protease and prevents HIV protease from functioning properly so that there is no maturation of HIV virions,
and this prevents other cells from being infected with HIV and the spread of HIV among human cells.

Case study: PFK for product inhibition and allosteric activators // Haemoglobin
Metabolic pathway:
a) a series of reactions each catalysed by an enzyme
b) product of 1 reaction = substrate for another reaction
c) End product accumulates & act as an allosteric inhibitor, but when end-product is used up, inhibition
is lifted & production is turned back on (example of negative feedback)
Allosteric activators:
a) Enables an enzyme to perform its function
b) Binds to enzyme at its allosteric sites & changes conformation of enzyme so enzyme can bind to
substrate & catalyse product formation

Allosteric activators such as AMP and ADP bind to the allosteric site as to facilitate the formation of the R
state by inducing structural changes in the enzyme.

Similarly, inhibitors such as ATP and PEP bind to the same allosteric site and causing a change in the
enzyme's 3D shape/ facilitate the formation of the T state, thereby inhibiting enzyme activity.

PFK is also inhibited by low pH. pH falls when muscle is functioning anaerobically and producing excessive
quantities of lactic acid. This inhibitory effect serves to protect the muscle from damage that would result
from the accumulation of too much acid.

The binding of oxygen (allosteric activator + substrate) to one subunit induces a conformational change in
that subunit that interacts with the remaining active sites to enhance their oxygen affinity.

With reference to fig.1.1, explain the effect of increasing temperature on the enzyme
activity for the first 10 minutes of the reaction.
Reaction at 70o had the highest concentration of glucose formed per unit time (28 au/ min)
Increase in kinetic energy of enzyme and substrate molecules result in an increase in frequency of effective
collisions between enzyme and substrate.

Increase in molecules with sufficient energy to overcome activation energy results in an increase in
enzyme-substrate complex formed per unit time.

Explain why an increase in substrate concentration at low substrate concentration

increases the initial rate of reaction but an increase at high substrate concentration does
At low substrate concentrations, there are more active sites available. Increase in substrate concentration
will result in increase in frequency of effective collisions between enzyme and substrate molecules, which in
turn lead to an increased rate of enzyme-substrate complex formation.

At high substrate concentration, there is saturation of active sites at any one time.

Substrate concentration is a limiting factor at low substrate concentration but not a limiting factor at high
substrate concentration.

How is the structure of RNA polymerase adapted to its role?

RNA polymerase has a specific active site complementary in shape and charge to the ribonucleotides.

R groups of catalytic amino acids capable of catalysing formation of phosphodiester bonds between