Structural Biochemistry/Enzyme/Competitive Inhibitor

Competitive Inhibitors belong to the category of enzymes known as reversible inhibitors. Reversible inhibitors dissociate the enzyme-inhibitor complex as soon as possible. They are inhibitors that bind directly to the active site of an enzyme, however they can also bind between an enzyme and a substrate. The competitive inhibitor competes with the substrate to bind to the enzyme. A competitive inhibitor mimics the substrate, competing for the active site. A competitive inhibitor can be overcome by increasing the substrate concentration. The excess amount of substrate can negate the competitive inhibitor and the maximum velocity is ultimately unaffected. Competitive inhibitors are effective because oftentimes they are structural analogs of the substrate that the enzyme binds, that is why the inhibitor is able to bind to the active site of the enzyme and compete with the original substrate.

Competitive inhibitors bind to the active sites of an enzyme and decrease the amount of binding of the substrate or ligand to enzyme. The result is that the Km is increased and Vmax remains the same. Ultimately, the chemical reaction can be reversed by increasing concentration of substrate.

E + S → ES → E + P 　　　vs. EI − (S comes in and replace I) → ES → E + P

(an equilibrium reaction also occurs at the same time: E + I ⇌ EI)

where E is the enzyme, I is the inhibitor, ES is the enzyme-substrate complex, P is the product, and EI is the enzyme-inhibitor complex.

Note: All the arrows also represents the reversible reactions. However, the reaction tends to proceed towards the right in the formation of products. Notice that there isn't any ESI formation. This means that the enzyme cannot bind to both the substrate and the inhibitor.




 * Competitive inhibition is reversible when enough substrate is present, meaning that the amount of inhibition depends on the concentration of inhibitor as well as the concentration of the substrates.
 * This inhibition makes the maximum rate of enzyme kinetics unchanged, but KM, Michaelis constant*, increases.

The Michaelis constant (Km) is:

1) the yield of the substrate concentration at the velocity of the half of the maximum velocity, or

2) half of the substrates at the maximum velocity. [Which is not true in most of the cases, but it is beyond of our discussion in this class and will be discussed in graduate school's courses]



The picture shows a double-reciprocal plot of V0 and [S]. The x-intercept is equal to -1/Km while the y-intercept is 1/Vmax. The slope of the line is Km/Vmax. Thus, the plot shows that there is an increase in Km and no change in Vmax.

The Michaelis-Menten equation becomes Vo= Vmax[S]/ aKm + [S] Where a = 1 + [I]/KI and   KI = [E][I]/[EI]

Competitive inhibitors can also be used to find the active site of an enzyme. N-(phosphonacetyl)-L-asparate, also known as PALA, is a competitive inhibitor which blocks the binding of Aspartate transcarbanoylase to its active site. PALA stabilizes the R state.

Penicillin-based antibacterials are examples of materials that compete at the active site of an enzyme in an inhibitory fashion. In general, penicillin drugs are used medicinally as antibiotics in the treatment of many bacterial infections; moreover, penicillin drugs derive their antibacterial action due to the fact that they bind irreversibly to bacterial glycopeptide transpeptidase. When unchecked, bacterial infections proliferate, in part, because of their ability to construct cell walls. A key enzyme in the synthesis of bacterial cell walls is transpeptidase. This enzyme plays a critical role in the cross-linking of peptidoglycan strands. Penicillin drugs inhibit the ability of transpeptidase to perform this crucial task. Without the cell wall, bacteria are unable to proliferate, which means the bacteria are essentially destroyed. Mechanistically, in the initial stage of the inhibitory action of penicillin-based drugs, the bond between the carbonyl carbon and the nitrogen atom in the β-lactam ring of the penicillin cleaves. The resulting electrophile is attacked by the newly formed alkoxide ion on the serine residue to form an ester, which results in the final product: a penicilloyl-enzyme complex between glycopeptide transpeptidase and penicillin. It is noteworthy to mention that this complex is stable indefinitely.



Introduction of Penicillin
Penicillin is an antibiotic agent that was earliest discovered and used widely. This antibiotic agent was derived from the Penicillium mold. Since antibiotics released by fungi and bacteria act as a natural substances that inhibits other organisms, it is then a chemical warfare on a microscopic scale. Penicillian is used to treat variety of infections and micro-organism.

History of Penicillin
Penicillin was first noticed in 1896 by Ernest Duchesne, a French medical student. Then in 1928, it was re-discovered by Alexander Fleming, a bacteriologist, who worked the London's St. Mary's Hospital. During Fleming's work at the hospital, he noticed a Staphylococcus plate culture that was contaminated by a blue-green mold. At the mean time, the colonies of bacteria that was next to the mold were dissolved. With much curiosity, Fleming began to grew the mold in a pure culture; as a result, he found that it formed a substance that killed a number of disease-causing bacteria. From the observations and experiments, Fleming named the substance penicillin, and Fleming published the results in 1929.

In 1938, Howard Florey, Ernst Chain and Norman Heatley continued the research of penicillin at Oxford University. During that period of time, the three scientist and their staff developed methods for growing, extracting, and purifying penicillin to prove its value as a drug.

Then during the World War II (1939–1945) period, penicillin became very useful. In 1941, the research and production of penicillin moved to the United States. It was to protect the progress and production of penicillin from bombings in England. More and more work began on the growing of mold to make penicillin in large quantities for thousands of soldiers. When the amount of people dying began to grow, the interest in penicillin also grew in laboratories, universities, and drug companies. Scientist at that time knew that they were in a race against death, and since this infection was able to kill a wounded soldier through a small wound.

Penicillin as an Inhibitor
'''Penicillin kills bacteria by interfering with the ability to synthesize cell wall. The bacteria lengthen, but cannot divide. Eventually the weak cell wall ruptures.'''

Penicillin irreversibly blocks bacterial cell wall synthesis by inhibiting the formation of peptidoglycan cross-links. Penicillin covalently binds to the enzyme transpeptidase that links the peptidoglycan molecules in bacteria, it inhibits the molecule so that it cannot react any further and cell wall cannot be further synthesized. The cell wall of the bacterium is weakened even further because the build-up of peptidoglycan precursors triggers bacterial cell wall hydrolysis and autolysins, and destroys pre-existing peptidoglycan. Penicillin makes a great inhibitor because of its four membered beta lactam ring, which makes it especially reactive. Penicillin acts as a suicide inhibitor by binding with the transpeptidase enzyme it inactivates itself.

Gram positive bacteria are the most sensitive and susceptible to penicillin because Gram positive bacteria only have murein (peptidoglycan) layer. Gram negative bacteria are usually more resistant to penicillin because they have multiple membrane layers, which allows them to still retain a cell wall even though they have lost their murein layer to penicillin. Penicillin can be used to treat Gram positive bacteria such as streptococcus pneumoniae, staphylococcus aureus, enterococcus, clostridium tetani, and listeria monocytogenes. Penicillin cannot be used to treat gram negative bacteria such as neisseria gonorrhoeae, neisseria meningitidis, pseudomonas, legionella, escherichia coli, helicobacter pylori (stomach ucler), borrelia burgdorfeli (lyme disease), treponema pallidium (syphilus), and chlamydia trachomatis.

Some bacteria have developed resistance to beta-lactams. These bacteria contain beta-lactamases, a broad class of enzymes with a serine residue that cleaves the reactive beta lactam ring through an acyl-enzyme intermediate. Augmentin, a drug that contains both a beta-lactam (typically amoxicillin) and clavulanic acid (a beta-lactamase inhibitor), is often prescribed to overcome drug resistant strains. Clavulanic acid works by competitive inhibition.

Benzylpenicillin
Benzylpenicillin, commonly known as penicillin G, is known as the gold standard penicillin. It is given by a method of non-oral administration (parentally) because it is unstable in the hydrochloric acid of the stomach. Because the drug is given parenterally, tissue concentrations of penicillin G can be achieved in larger levels than is possible with other types of penicillin, like phenoxymethylpenicillin. These higher concentrations become increased antibacterial levels or activities.

Uses for benzylpenicillin include:


 * Cellulitis
 * Bacterial endocarditis
 * Gonorrhea
 * Meningitis
 * aspiration pneumonia, lung abscess
 * Community-acquired pneumonia
 * Syphilis
 * Septicemia in children

Phenoxymethylpenicillin/penicillin V
Phenoxymethylpenicillin is the orally active form of penicillin. It is less frequently used than benzylpenicillin, and is mostly used in conditions when high tissue is not required. However, it is the first choice when it comes to treating odontogenic (relating to the teeth) infections.

Procaine
Procaine benzylpenicillin (rINN), also known as procaine penicillin, is a combination of benzylpenicillin with the local anaesthetic agent procaine. It is absorbed into the circulation by means of deep intramuscular injection. It is used when prolonged low concentrations of benzylpenicillin are needed, and mostly used in veterinary environments as well as dental offices. The common trade name for procaine is Novocain which is typically administered to patients at the dentist office before undergoing minor surgery. It is metabolized in the blood plasma by an enzyme called pseudocholinesterase. Procaine replaced cocaine as a local anesthetic due to the severe side effects caused by cocaine.

Benzathine
Benzathine benzylpenicillin (rINN), also known as benzathine penicillin, is absorbed into the circulation slowly by intramuscular injection like procaine penicillin, but then it is hydrolysed to benzylpenicillin in vivo. It is the number one drug choice when prolonged low concentrations are required and appropriate. It allows for antibiotic action to be prolonged over two to four weeks following just one dose.

Other examples of penicillin include amoxicillin, ampicillin, methicillin, oxacillin, and temocillin. Amoxcillin and ampicillin are the most common penicillins that are prescribed by doctors because they are used to treat common infections such as throat infections.

Side effects
Just like any other drug on the market, penecillin may cause unpleasant side effects for patients taking it. Some of the side effects are commonly found on patients using penicillin are: diarrhea, hypersensitivity, nausea, rash, neurotoxicity urticaria, seizures. Pain and inflammation at the injection site is also common for the partenterally administered pencillin types. 3% to 10% of the population are allergic to penicillin. Usually when allergic to one type of penicillin, you are allergic to the entire family of penicillin antibiotics. The problems mentioned about diarrhea and nausea tend to go away for most patients after a few doses of the drug. If further symptoms occur, however, the drug should not be taken anymore. Physicians say that any drug that interferes with cellular growth, in this case the cell wall, can have severe side effects on a large population of patients.

Penicillin allergy
Penicillin and related antibiotics can cause an allergic reaction in some people. Although, not all adverse reactions to penicillin are a sign of an allergic reaction. True allergic reactions involve the immune system and can cause signs and symptoms that range from an annoying rash to a life-threatening reaction or anaphylaxis with low blood pressure and trouble breathing. β-lactam antibiotic can end up causing allergic reactions to about 10% of the patients. However, even though penicillin are very commonly reported in allergy cases, but less than 20% of those reports are truly allergic. But it is definitely a drug that can cause major, severe reactions.

It isn't clear why some people develop penicillin allergy while others don't. Treating an allergic reaction may require taking medications or emergency care in serious cases.