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, and 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 structure and amino acids sequence, and different enzyme parameters (Km, Vmax,

kcat); making the metabolism better.

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

They are produced by different genes that vary 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 and it cannot cross the plasma membrane out

of cells 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.

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.

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

mM).

High Km 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.

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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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 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.

‘convert excess glucose in 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.

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 isozyme is

characteristic of that from heart

tissue, and the all M isozyme is typically found in skeletal muscle and liver.

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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+.

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

functions anaerobically and catalyzes the conversion of pyruvate into lactate, and this lactate

moves in the blood until it reaches the heart. Now, the all H enzyme (H4) functions aerobically

and catalyzes the reverse reaction (Lactate oxidation) producing pyruvate again, and then, it

undergoes aerobic respiration, producing large amounts of ATP. (See picture in previous page)

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.

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This 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. And 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).

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 (means low affinity of substrate).

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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 be 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.

Note that; 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.

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.

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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.

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. And that is because scientists found that the transition

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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.

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).

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C. Heavy Metals

Heavy-metal toxicity is caused by 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.

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, & 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”