Neaam Al-Bahadili

Nebal Al-Gallab

22

...

Mamon & Nafith

AbuTAarbudh

Ahram

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Enzyme Regulation

Enzymes are used to catalyze (speed up) reactions within the body, they are highly controlled and

regulated; to help in maintaining the body's equilibrium.

Enzymes are regulated in several modes/mechanisms:

1. Isoenzymes

2. Regulation of enzymatic activity

a. Inhibitors

b. Conformational changes

Allosteric

Modulators

Reversible covalent modification

Irreversible covalent modification

3. Regulation of enzyme amount

4. Location (Compartmentalization and complexion of enzymes); storing enzymes in specific

compartments

5. Non-specific regulation; targets all enzymes in the same manner

1- Isoenzymes (Isozymes)

Isoenzymes are two or more enzymes with identical function (catalyzing the same reaction), but with

different structures and amino acid sequences, and different enzyme parameters (Km, Vmax, kcat);

which makes the metabolism better.

They act on the same substrate(s) producing the same product(s).

They are produced by different genes that differ only slightly.

Various isozymes are present in different tissues of the body/different distribution, according to

the particular metabolic needs of the tissue.

They can be regulated differently.

They have different catalytic activities.

Examples of isozymes: Hexokinases and Lactate dehydrogenases (LDH).

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Hexokinase and Glucokinase

These enzymes catalyze the same

reaction converting glucose to glucose-6-

phosphate. However, they are expressed

in different tissues and have different

kinetic and regulatory properties.

Hexokinase type I is the predominant form in muscles and red blood cells, to produce energy

(Glucose is the only energy source for RBCs), and it is inhibited by glucose-6-phosphate.

Hexokinase type IV (Glucokinase) is the predominant form in the liver and pancreas, to store

glucose and balance glucose level in the blood. It is not inhibited by the production of

glucose-6-phosphate.

Once glucose is phosphorylated, it is trapped inside the cell and it cannot cross out the plasma

membrane; due to the negative charges it carries. Therefore, it is important for the liver to have an

enzyme with lower efficiency to phosphorylate glucose; in order to provide it to other organs like

muscles.

In contrast, it is important for muscles to have an enzyme with high efficiency for phosphorylating

glucose; in order to trap it for energy production.

The Km value of hexokinase for glucose is low (0.1 mM), but it is high for glucokinase (10

mM).

High Km value of hepatic glucokinase promotes storage of glucose.

When the substrate concentration is below the Km value, the enzyme is not effective. However,

when the substrate concentration is above the Km value, the enzyme is highly effective.

Remember: Hexokinase is inhibited by glucose-6-phosphate, but Glucokinase is not. Because

muscles and RBCs do not consume all glucose in blood, however, the liver can use excess glucose

in glycogen for storage.

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Hexokinase I Hexokinase IV (Glucokinase)

Substrate Several Hexoses Glucose

Product Glucose-6-Phosphate Glucose-6-Phosphate

Site of Action RBCs and Muscles Concentrated in the Liver and

Pancreas

Activity (related to Km) Remains active even at low glucose

levels.

Only works at high glucose levels

when a large quantity of glucose is

present in the liver.

Km value for glucose Low

(High affinity for glucose)

High

(Low affinity for glucose)

Feedback Inhibition Inhibited by glucose 6 phosphate

Has no direct feedback inhibition.

‘Converts excess glucose to

glycogen for storage’.

Lactate dehydrogenases (LDH)

LDH is an enzyme found in nearly all living cells. It catalyzes the conversion of lactate to pyruvic acid

and back, as it converts NAD⁺ to NADH and back.

Notes:

- The normal fasting blood sugar level ≈ 5 mM

-RBCs: when blood glucose falls below its normal fasting level (≈ 5 mM), RBCs could still

phosphorylate glucose at rates near Vmax, because Km value is below 5 mM.

-Liver: rate of phosphorylation increases above fasting levels (after a high carbohydrate meal).

-Pancreas: works as a sensor

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Structure: It is a tetramer; an enzyme composed of a combination of 4 protein subunits: either H

(heart) or M (skeletal muscle). These subunits combine in various ways leading to 5 distinct

isozymes (LDH1-5) with different combinations of the M and H subunits; 4H, 4M, and the three

mixed tetramers (3H1M, 2H2M, 1H3M). These five isozymes are enzymatically similar but

show different tissue distribution; all H isozymes have characteristics of that from heart tissue,

and the all M isozymes are typically found in skeletal muscle and liver.

To understand this mechanism, you have to remember the metabolism of glucose. The breakdown of

glucose to provide energy begins with glycolysis. First, glucose enters the cytosol of the cell, next,

glucose is converted into two or three-carbon molecules of pyruvate through a series of different

reactions producing ATP. So, glucose will be completely degraded to pyruvate.

Then, the pyruvate takes one of two pathways; either aerobic respiration in the presence of oxygen, or

anaerobic respiration in the absence of oxygen where pyruvic acid is reduced to lactic acid and NADH

is oxidized to NAD+.

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Muscles can function anaerobically, but heart tissues cannot. Therefore, the all M isozymes (M4)

function anaerobically and catalyze the conversion of pyruvate into lactate, and this lactate moves in the

blood until it reaches the heart. Now, the all H isozymes (H4) function aerobically and catalyze the

reverse reaction (Lactate oxidation) producing pyruvate again, and then, it undergoes aerobic

respiration, producing large amounts of ATP.

H4 LDH has low Km for pyruvate and is inhibited by high levels of pyruvate. However, M4 LDH has

high Km for pyruvate and is not inhibited by pyruvate.

2- Regulation of enzymatic activity: Inhibition

Enzyme inhibition can be:

Irreversible: an irreversible inhibitor dissociates very slowly from its target enzyme

because it has become tightly bound to the enzyme, mainly covalently.

The kinetic effect of irreversible inhibitors is to decrease the concentration of active

enzyme.

- Metals, toxins, poison, pharmaceutical drugs are irreversible inhibitors.

Reversible: it is characterized by a rapid dissociation of the enzyme-inhibitor complex.

Usually these inhibitors bind to enzymes by non-covalent forces and the inhibitor

maintains a reversible equilibrium with the enzyme. They can be competitive or

noncompetitive inhibitors.

- All physiological inhibitors in the body are reversible.

Competitive Inhibitors

In competitive inhibition, the inhibitor competes with the substrate for the active site because

of their molecular similarity. It will block the enzyme's active site (it will occupy the same space

as the natural substrate, blocking it from being catalyzed).

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Because increasing the amount of substrate can overcome the inhibition, Vmax can be reached

in the presence of a competitive inhibitor. And the enzyme will have the same Vmax as in the

absence of an inhibitor, but the value of Km (which is an indication of the affinity) will be

increased (which means a low affinity of substrate).

Non-Competitive Inhibition

A non-competitive inhibitor will bind to the enzyme somewhere other than the active site of

the enzyme; an allosteric site. Even if the substrate can bind to the enzyme-inhibitor complex, it

will not proceed to form product.

Unlike competitive inhibition, noncompetitive inhibition cannot overcome by increasing the

substrate concentration, because the inhibitor is not in direct competition with the substrate.

The value of Vmax is decreased, by reducing the number of active enzyme molecules. While

the value of Km is unchanged.

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Irreversible Inhibitors (Mechanism-based inhibitors)

Mechanism-based inhibitors mimic or participate in an intermediate step of the catalytic

reaction. The kinetic effect of irreversible inhibitors is to decrease the concentration of the active

enzyme.

The term includes:

A. Covalent inhibitors

B. Transition state analogs

C. Heavy metals

A. Covalent inhibitors

Such inhibitors directly form covalent or extremely tight bonds with

the active site amino acids. So, the enzyme will no longer be active.

Example: DFP, It is a lethal organophosphorus compound, and a

prototype for the nerve gas sarin that is considered a war gas, and the

insecticides malathion & parathion. DFP inhibits acetylcholinesterase

preventing the degradation of the neurotransmitter acetylcholine into

choline and acetate, which means that the NT will always be active and

there will always be action in the CNS, eventually it will cause paralysis

to the human bodies and then they die. DFP also inhibits other enzymes

that use serine (ex. serine proteases), but the inhibition is not as lethal.

Note: Competitive inhibitors do not alter the structure of the enzyme, while non-competitive

inhibitors alter the structure of the enzyme in such a way that the substrate may bind to the active

site but products will not be formed.

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Another example is Aspirin (acetylsalicylic acid): as we took before, Aspirin is responsible for

the inhibition of prostaglandins. Aspirin results in covalent acetylation of the active site serine in

the enzyme prostaglandin endoperoxide synthase (cyclooxygenase COX), so the enzyme will be

inhibited and prostaglandins will no longer be produced.

B. Transition-State Analogs & Compounds (Suicide Inhibitors)

They are extremely potent inhibitors, which means they bind

more tightly. They are chemical compounds with a chemical

structure that resembles the transition state of a substrate molecule

in an enzyme-catalyzed chemical reaction. That is because

scientists found that the transition state of the substrate binds more

strongly to the active site than the substrate itself. They are

irreversible inhibitors, because it is very hard to break their bond

with the active site.

One example is the drug used in cancer chemotherapy;

Methotrexate. It is a structural analog of tetrahydrofolate (a

coenzyme for the enzyme dihydrofolate reductase) which plays a

role in the biosynthesis of nucleotides.

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How is it used in treating cancer? Cancer cells keep on dividing uncontrollably, and for the cell

to divide, it needs to synthesize DNA, and so it needs a lot of nucleotides. One of the enzymes

that catalyze the synthesis of nucleotides is dihydrofolate reductase, this enzyme needs a cofactor

tetrahydrofolate which is reduced to dihydrofolate after producing the nucleotide. Now the

dihydrofolate needs to be converted to tetrahydrofolate again using dihydrofolate reductase

enzyme.

Here comes the role of Methotrexate; it binds to dihydrofolate reductase 1000-fold more

tightly than the natural substrate and inhibits nucleotide base synthesis. Methotroxate is similar

to dihydrofolate, when it binds the enzyme, it will prevent the synthesis of tetrahydrofolate, and

therefore, it will prevent cell division.

Another example is Penicillin; It is a transition-state

analog to glycopeptidyl transpeptidase, which is a bacterial

enzyme that cross-links the peptidoglycan chains to form

rigid cell walls. The peptide bond in the β-lactam ring of

penicillin looks like the natural transition-state complex. The

active site serine attacks the highly strained β -lactam ring,

resulting in opening of the lactam. This reaction leads to

irreversible covalent modification of the enzyme. (See

following figure).

C. Heavy Metals

Heavy-metal toxicity is caused by the tight binding of a metal such as mercury (Hg), lead (Pb),

aluminum (Al), or iron (Fe) to a functional group in an enzyme.

Mercury binds to many enzymes, often at reactive sulfhydryl groups (thiol-SH) in the active

site. It has been difficult to determine which of the inhibited enzymes is responsible for mercury

toxicity.

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Lead provides an example of a metal that inhibits through replacing the normal functional

metal in an enzyme such as calcium, iron, or zinc by irreversible mechanism. Its developmental

& neurologic toxicity may be caused by its ability to replace Ca+2 in several regulatory proteins

that are important in the central nervous system and other tissues.

Abzymes

Abzymes (from antibody and enzyme) are antibodies that catalyze specific chemical reactions

i.e., function as enzymes. It is an antibody that is produced against a transition-state analog &

that has catalytic activity similar to that of a naturally occurring enzyme.

An abzyme is created by injecting a host animal with a transition-state analogue. The host

animal makes antibodies to the foreign molecule, and these antibodies have specific binding

points that mimic the active site of the enzyme surrounding a transition state.

“You are not studying to pass the exam ..

You are studying for the day when you are

the only thing between the patient and the grave”