Enzymes III - humsc.net
Transcript of Enzymes III - humsc.net
“Enzymes III” “Regulation – Part I (Isozymes & Inhibitors)”
Isoenzymes - Isozymes
In this topic, we will learn about the following terms:
❖ Expression of isoenzymes
❖ Regulation of enzymatic activity:
✓ Inhibitors
✓ Conformational changes
▪ Allostery
▪ Modulation
• Small vs. large, covalent vs. non-covalent, reversible vs. irreversible
❖ Regulation of enzyme amount
❖ Location (Compartmentalization and complexing of enzymes)
❖ Non-specific regulation
Isoenzymes (Isozymes)
Isoenzymes are enzymes that can act on the same substrate(s) producing the same product(s).
They are produced by different genes that vary only slightly.
Often, various isozymes are present in different tissues of the body.
They can be regulated (Activation, Inhibition) differently.
They can have different catalytic activities.
✓ The arrows’ weights indicate the efficiency.
✓ The black arrow indicates activation, the (X) symbol indicates inhibition.
Aerobic vs. anaerobic metabolism
Aerobic metabolism is the way your body creates energy
through the combustion of carbohydrates, amino acids,
and fats in the presence of oxygen.
✓ Aerobic metabolism is used for the sustained
production of energy for exercise and other body
functions. Examples of exercises that use aerobic
metabolism include walking, running, or cycling
with sustained effort.
Anaerobic metabolism is the creation of energy through
the combustion of carbohydrates in the absence of oxygen.
✓ This occurs when your lungs cannot put enough
oxygen into the bloodstream to keep up with the
demands of your muscles for energy. It generally is
used only for short bursts of activity, such as when
you sprint when running or cycling or when you are
lifting heavy weights.
Your body will often switch between aerobic and anaerobic metabolism during sports and exercise
activities that require short bursts of sprints as well as sustained jogging, such as in soccer,
tennis, and basketball.
Aerobic metabolism Anaerobic metabolism
Speed Slower Faster
Production of ATP (Energy) More ATP Less ATP
Lactate dehydrogenases (LDH)
LDH is a tetrameric (4 subunits) enzyme composed of a combination of one or two protein
subunits: H (heart) and 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.
The all H isozyme is characteristic of that from heart tissue, and the all M isozyme is typically
found in skeletal muscle and liver.
Although the five isoforms catalyze the same reaction, they differ in their primary structure,
kinetic properties, tissue distribution, affinity to the substrate, regulation, and isoelectric point (the
pH at which a particular molecule carries no net electrical charge).
The M subunit has a net charge of (-6) and higher affinity towards pyruvate, thus converting
pyruvate to lactate and NADH to NAD+.
The H subunit has a net charge of (+1) and a higher affinity towards lactate, resulting in a
preferential conversion of lactate to pyruvate and NAD+ to NADH.
Logic behind tissue distribution:
Isoenzyme Structure Present in Elevated in
LDH1 (H4) Myocardium myocardial infarction
LDH2 (H3M
1) RBC
LDH3 (H2M
2) Lungs
LDH4 (H1M
3) Kidney
LDH5 (M4) Skeletal muscle, Liver
Skeletal muscle and liver diseases
***You are supposed to know the first & last ones only (For now).
Muscles can function anaerobically, but heart tissues cannot.
Whereas the all M isozyme (M4) functions anaerobically and catalyzes the reduction of pyruvate
into lactate, the all H enzyme (H4) functions aerobically and catalyzes the reverse reaction.
The pathway of this reaction as follows:
1. Glucose is being converted into Pyruvate.
2. The skeletal muscles will use the ATP in the tissue, then they will use another source
(Creatine phosphate), after that they will use the anaerobic metabolism.
3. In the skeletal muscles, Pyruvate is being converted
into lactate, then the lactate will move with the blood
to reach the heart.
✓ Efficiency for converting pyruvate to lactate is
high.
4. The heart will convert lactate into pyruvate. Since the
heart cannot work anaerobically, the pyruvate will
enter the aerobic metabolism.
✓ If [Pyruvate] is high, the lactate will stop from
producing.
✓ Use the following diagram to understand the
concept perfectly.
Regulation of LDH:
H4 LDH has a low Km for lactate, high Km for pyruvate, and is inhibited by high levels of pyruvate.
✓ The H4 isoenzyme favors (lactate to pyruvate).
The M4 LDH enzyme has a high Km for pyruvate and is not inhibited by pyruvate.
✓ M4 LDH is always active even at high levels of pyruvate ensuring that pyruvate is always
funneled to anaerobic metabolism.
Hexokinase vs glucokinase
Hexokinase and glucokinase (hexokinase IV) are allosteric isozymes that catalyze:
Glucose → Glucose-6-Phosphate
Glucokinase is a liver (and pancreatic) enzyme, whereas hexokinase is an RBC (and skeletal
muscle) enzyme.
❖ The purpose of liver glucose is to balance glucose level in the blood.
✓ Liver stores glucose in the form of Glycogen. Hence, balancing glucose level in the
blood.
❖ The purpose of RBC glucose is to produce energy.
When the glucose concentration in blood is high, the storage of it as glycogen in the liver will
increase. Otherwise, the glycogen will be converted into glucose.
The two enzymes are considered as isozymes because they catalyze the same reaction with
different places, and other reasons will be discussed next.
Biological significance:
Note: once glucose is phosphorylated, it cannot cross plasma membrane out of cells.
❖ Liver: low efficiency enzyme to provide glucose to other organs.
❖ RBC and skeletal muscles: high efficiency enzyme to trap glucose.
Hexokinase
Glucokinase
In other word:
❖ In the liver, glucose is not converted directly to G-6-P, the liver will make sure that there is
not demand for glucose, and then convert & store it as glycogen. That is why the enzyme in
the liver is lowly efficient.
❖ In the RBCs, glucose is directly converted to G-6-P to trap glucose and produce energy.
That is why the enzyme in the RBCs is highly efficient.
Regulation of hexokinase and glucokinase
Consider the following graphs to study the regulations of the two enzymes:
✓ When the [Glucose] is high, RBCs, skeletal muscles, and the liver are all fine. The glucose
will be converted to glycogen in the liver, and it will be used to produce energy in the RBCs
and skeletal muscles.
✓ Notice how the two enzymes are highly active when the [Glucose] is high.
✓ Notice how the Vmax (Efficiency) for Glucokinase (Liver) is higher than hexokinase (RBCs).
✓ Notice the fasting blood glucose and how the activity of glucokinase is low.
✓ The activity of hexokinase at the level of fasting blood glucose is maximal (Produce
energy).
✓ Notice how the two enzymes are regulated differently (Isozymes).
Note Vmax and KM values (low - 0.1 mM for hexokinase and (high - 10 mM for glucokinase)
Regulation:
Hexokinase is inhibited by glucose-6-phosphate, but glucokinase is not.
Glucokinase is activated by insulin and inhibited by glucagon.
Significance:
At fasting state, glucose is not stored.
At well-fed state, RBCs and skeletal muscles do not consume all glucose in blood and liver can
convert excess glucose in glycogen for storage.
Regulation of enzymatic activity - Inhibitors
Enzyme inhibition can be either reversible or irreversible.
❖ An irreversible inhibitor is tightly bound (e.g. covalently) to the enzyme.
✓ Lower concentration of active enzyme.
✓ They are used in drugs & treatments.
❖ Reversible inhibitors rapidly dissociate from enzymes (e.g. non-covalent binding).
✓ Competitive, noncompetitive, or uncompetitive inhibition.
✓ All physiological inhibitors are reversible.
Competitive inhibition
Competitive inhibitors compete with the substrate for the active site.
❖ Increasing substrate can overcome inhibition.
❖ Same Vmax, but higher KM.
✓ Notice how the competitive inhibitors increases KM.
Noncompetitive inhibition
Noncompetitive inhibitors bind E or ES complex at a site other than the catalytic site.
Substrate can bind to the enzyme-inhibitor complex, but ESI cannot form a product.
❖ Lower Vmax, but same KM.
Uncompetitive inhibition
Uncompetitive inhibitors bind to the enzyme-substrate complex only reducing both Vmax and KM.
Notice how both Vmax & KM are reduced:
Mechanism-based inhibitors (irreversible inhibitors)
Mechanism-based inhibitors mimic or participate in an intermediate step of the catalytic reaction.
They include:
❖ Covalent inhibitors.
❖ Transition state analogs.
❖ Heavy metals.
Irreversible inhibitors decrease the concentration of active enzyme.
Covalent inhibitors
They form covalent or extremely tight bonds with active site amino acids.
❖ Example: diisopropyl fluorophosphate (DFP) is an organophosphate:
✓ Used as the nerve gas sarin (In wars which can cause cramps – تشنجات).
❖ The insecticides malathion & parathion.
❖ DFP inhibits acetylcholinesterase preventing the degradation of the neurotransmitter
acetylcholine.
✓ Acetylcholinesterase make degradation for acetylcholine, so the nerve can relax.
DFP also inhibits other enzymes that use serine (ex. serine proteases), but not lethal – ليست
.قاتلة
Another good example is Aspirin:
Aspirin (acetylsalicylic acid) acetylates an active site serine of cyclooxygenase.
❖ Cyclooxygenase is the enzyme that will make eicosanoids (20 Carbon lipids molecules
produced by arachidonic acid).
❖ Eicosanoids are responsible for:
✓ Blood clotting.
✓ Inflammation.
✓ Sensation (Infection) of fever.
❖ Aspirin inhibits cyclooxygenase.
Aspirin resembles a portion of the prostaglandin precursor that is a physiologic substrate for the
enzyme.
Substrate and transition-State analogs (Suicide inhibitors)
They bind more tightly than substrates.
Drugs cannot be designed that precisely mimic the transition state! (Highly unstable structure).
Example 1: Methotrexate:
Methotrexate is a synthetic inhibitor used to treat cancer.
It is a structural analog of folate, a substrate for the enzyme dihydrofolate reductase, and a
coenzyme for thymidylate kinase, both of which are responsible for the synthesis of nucleotides.
It binds to dihydrofolate reductase 1000-fold more tightly than the natural substrate and inhibits
nucleotide base synthesis.
✓ Tetrahydrofolate is the substrate. Notice how it looks like Methotrexate.
✓ Methotrexate inhibits DHFR.
Example 2: Penicillin:
It is a transition-state analog to glycopeptidyl transpeptidase, which is
required for synthesis of the bacteria cell wall.
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.
✓ Penicillin is a suicide inhibitor because it kills itself and the enzyme (Nothing is working).
Heavy Metals
Mercury (Hg), lead (Pb), aluminum (Al), or iron (Fe) result in tight binding to a functional group in
an enzyme.
❖ Nonspecific inhibition at high doses.
✓ At high doses, the heavy metals will bind randomly, not to specific group (Non-
specific inhibition).
Mercury binds to reactive sulfhydryl groups away from the active site and affect the binding of
substrates.
❖ Unknown enzymes in mercury toxicity.
Lead replaces 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.
A summary for reversible inhibitions:
✓ Notice how the slope increases in the Competitive & Noncompetitive, but in the
uncompetitive it is constant.
Characteristics Competitive Noncompetitive
Shape of inhibitor Similar shape to the substrate Does not have a similar shape to the substrate
Binding to Enzyme Competes for and binds at the active site
binds away from the active site to change the shape of the enzyme and its activity
Reversibility Adding more substrate reverses the inhibitions
Not reversed by adding more substrate, but by a chemical change that removes the inhibitor
The End
Do not forget to answer the test bank.