Enzymes

Information Resources

Information Resources

1. Biological Chemistry: Textbook / A.L. Zagayko, L.M. Voronina, G.B. Kravchenko, K.V. Strel`chenko. – Kharkiv: NUPh; Original, 2011. – 58-72 p.

2. Training Journal for Licensed Exam “KROK-1”: Study Material in Biological Chemistry. – Kharkiv: NUPh, 2017. – 30-40 p.

3. Laboratory Manual on Biochemistry. Kharkiv: NUPh, 2017. - 36-42 p.

4. Nucleotide Metabolism: The Medical Biochemistry Page. Available on: https://themedicalbiochemistrypage.org/nucleotide-metabolism.php.

5. Enzyme Kinetics: The Medical Biochemistry Page. Available on: https://themedicalbiochemistrypage.org/enzyme-kinetics.php.

Enzymes are biological catalysts responsible for supporting almost all of the chemical reactions that maintain animal homeostasis.

The main enzyme qualities are great effectiveness and specificity.

Macromolecular components of almost all enzymes are composed of protein, except for a class of RNA known as ribozymes. Ribozymes are molecules of ribonucleic acid that catalyze reactions on the phosphodiester bond of other RNAs.

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Almost every significant life process is dependent on enzyme activity. Enzymes are found in all tissues and fluids of the body. Intracellular enzymes catalyze the reactions of metabolic pathways.

Plasma membrane enzymes regulate catalysis within the cells in response to extracellular signals.

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Enzymes of the blood circulatory system are responsible for some processes, regulating, for example, the blood clotting.

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Study of enzymes also has immense practical importance.

- In some diseases, especially inheritable genetic disorders, there may be a deficiency or even a total absence of one or more enzymes in the tissues.

- Abnormal conditions can also be caused by the excessive activity of a specific enzyme.

- Measurements of the activity of certain enzymes in the blood plasma, erythrocytes, or tissue samples are important in disease diagnosing.

- Enzymes have become important practical tools, not only in medicine but also in the pharmaceutical industry, in food processing, and in agriculture.

- Enzymes play a part even in everyday activities at home such as food preparation and cleaning.

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ENZYME NOMENCLATURE AND

CLASSIFICATION

Except for some of the originally studied enzymes such as pepsin, rennin or trypsin (so-called trivial nomenclature) most enzyme names end with "ase". The International Union of Biochemistry (I.U.B.) initiated standards of enzyme nomenclature which recommends that enzyme names indicate both the substrate acted upon and the type of reaction catalyzed.

According to this classification all enzymes have a unique number which contains from four numbers.

The first of them is the class:

1. Oxidoreductases: reactions of oxidation and reduction (dehydrogenases, peroxidases).

2. Transferases: transfer functional groups between donor and acceptor molecules: transglycosidases (monosaccharides), transphosphorylases (a phosphate group), transaminases (amino group), transmethylases (a methyl group), transacetylases (an acetyl group).

3.Hydrolases: splitting chemical bonds with water (hydrolases - esterases, nucleases, deaminases, amidases, and proteases).

4.Lyases: add water, ammonia or carbon dioxide across double bonds, or remove these elements to produce double bounds.Splitting different chemical bonds without wate (decarboxylases, dehydratases).

5.Isomerases: changing geometry or structure of a molecule (isomerases and mutases).

6.Ligases: Joining two molecules due to hydrolysis of pyrophosphate bond in ATP or other tri-phosphate (synthetases).

Nomenclature of enzymes

1) The first discovered enzymes were named according to their source: name of enzyme + suffix -in

Pepsin is found in the gastric juice (Greek pepsis = digestion).

2) Enzymes were named according to their substrate:

name of substrate + suffix –ase

Lipase catalyzes the hydrolysis of lipids.

Urease catalyzes the hydrolysis of urea.

3) In 1961 International Union of Biochemistry recommended that enzymes be systematically classified according to the general type of reaction they catalyze → 6 major classes.

Each enzyme has a EC number (four-digit number)

Lactate dehydrogenase has the EC number 1.1.1.27

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ENZYME STRUCTURE

With the exception of a small group of catalytic RNA molecules, most enzymes are proteins. Their catalytic activity depends upon the integrity of their native protein conformation.

Non-protein groups can be metal ions or a complex organic or metalloorganic molecule called a coenzyme (if they are bound with protein noncovalently) or a prosthetic group (if they are bound covalently).

A complete catalytically active enzyme together with its non-protein group is called a holoenzyme.

The protein part of such an enzyme is called apoenzyme or apoprotein and is catalyticaly inactive.

Usually coenzymes function as transient carriers of specific functional groups. The non-protein group can also take part in substrate binding, enzyme activity regulation or stabilization.

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Some Metal Ions, Coenzymes

and The Enzymes They Are Associated With

Active Site

The active site is a part of enzyme molecule where substrate binding and enzymatic reaction take place.

Active site comprises only a small portion of the overall enzyme structure. The active site is part of the conformation of the enzyme molecule arranged to create a special pocket or cleft which three-dimensional structure is complementary to the structure of the substrate.

https://www.researchgate.net/profile/Juergen_Markl/publication/235775655/figure/fig5/AS:269828506255360@1441343537409/Structure-of-the-molluscan-hemocyanin-subunit-A-Active-site-with-two-copper-ions.png

The enzyme and the substrate molecules “recognize” each other through this structural complementarity. The substrate binds to the enzyme due to relatively weak forces — H-bonds, ionic bonds (salt bridges), and van der Waals interactions between sterically complementary clusters of atoms.

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Allosteric Site

Some enzymes have an additional site - allosteric site which is separated from the active site and is important for their regulation. This site interacts with special molecules, they are called effectors, which can change the enzyme activity.

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MECHANISM OF ENZYME ACTION

The “Lock and Key” Hypothesis (Emil Fischer)

Mechanism for a single substrate enzyme catalyzed reaction. The enzyme (E) binds a substrate (S) and produces a product (P).

This analogy captures the essence of the specificity that exists between an enzyme and its substrate, but enzymes are not rigid templates like locks.

The “Induced Fit” Hypothesis (Daniel Koshland)

The binding of a substrate (S) by an enzyme is an interactive process. That is, the shape of the enzyme’s active site is actually modified upon binding S, in a process of dynamic recognition between enzyme and substrate aptly called "induced fit".

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ENZYME SPECIFICITY

The extraordinary ability of an enzyme to catalyze only one particular reaction is a quality known as specificity.

A lot of enzymes exhibit absolute specificity; that is, they will catalyze only one particular reaction. Other enzymes will be specific for a particular type of chemical bond or functional group. In general, there are three distinct types of specificity:

Absolute specificity - the enzyme will catalyze only one reaction.

Group specificity - the enzyme will act only on molecules that have specific functional groups, such as amino, phosphate or methyl groups.

Stereochemical specificity - the enzyme will act on a particular steric or optical isomer.

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Concentration of enzyme and substrate

Factors affecting catalytic activity of enzymes

For a given enzyme concentration, the rate of reaction increases with increasing substrate concentration up to a point, above which any further increase in substrate concentration produces no significant change in the reaction rate. This is because of the active site of the enzyme molecules at any given moment are virtually saturated with substrate. The enzyme/substrate complex has to dissociate before the active site is free to accommodate more substrate.

Provided that the substrate concentration is high and that temperature and pH are kept constant, the rate of reaction is proportional to the enzyme concentration.

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pH An enzyme possesses an array of ionizable side chains and prosthetic groups that not only determine its secondary and tertiary structures but may also be included in its active site. Further, the substrate itself often has ionizing groups, and one or another of the ionic forms may preferentially interact with the enzyme. Enzymes in general are active only in a limited pH range and most have a particular pH at which their catalytic activity is optimal.

Like most chemical reactions, the rates of enzyme-catalyzed reactions generally increase with increasing temperature. However, at temperatures above 50° to 60°C, enzymes typically show a decline in activity. Two effects are operating here: (a) the characteristic increase in reaction rate with temperature, and (b) thermal denaturation of protein structure at higher temperatures.

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Enzyme inhibitors are classified in several ways!

ENZYME INHIBITION

Reversible Versus Irreversible Inhibition

The inhibitor may interact either reversibly or irreversibly with the enzyme. Reversible inhibitors interact with the enzyme through noncovalent association/dissociation reactions.

In contrast, irreversible inhibitors usually cause stable, covalent alterations in the enzyme. That is, the consequence of irreversible inhibition is a decrease in the concentration of active enzyme.

Who are the people behind this idea?

The antibiotic penicillin exerts its effects by covalently reacting with an essential serine residue in the active site of glycoprotein peptidase, an enzyme that acts to crosslink the peptidoglycan chains during synthesis of bacterial cell walls

Suicide substrates are inhibitory substrate analogies designed so that, via normal catalytic action of the enzyme, a very reactive group is generated.

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Suicide substrates bind with specificity and high affinity to the enzyme active site; in their reactive form, they become covalently bound to the enzyme.

5-fluorouracil (which is converted in the body to 5F-dUMP) is a suicide inhibitor of thymidylate synthase, and prevents DNA synthesis in cancerous cells.

Reversible inhibitors fall into two major categories: competitive and noncompetitive.

Competitive inhibitors are characterized by the fact that the substrate and inhibitor compete for the same binding site on the enzyme, the active site or S-binding site.

Succinate dehydrogenase — a classic example of competitive inhibition. The enzyme succinate dehydrogenase (SDH) is competitively inhibited by malonate.

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Para-aminobenzoic acid (PABA) is essential growth factor for microorganisms. It forms part of the molecule of folic acid and is required for the synthesis of this vitamin. Mammals cannot synthesize folic acid, and PABA has no other known function. There is no evidence that it is a human dietary requirement. Sulphanilamides are chemical analogues of PABA, and express their antibacterial action by antagonizing PABA utilization.

Competitive acetylcholinesterase inhibitor Prozerin - inhibits the activity of acetylcholinesterase, causes accumulation of acetylcholine.

In noncompetitive inhibition, a molecule binds to an enzyme somewhere other than the active site. This changes the enzyme's three-dimensional structure so that its active site can still bind substrate with the usual affinity, but is no longer in the optimal arrangement to stabilize the transition state and catalyze the reation.

Examples of non-competitive inhibitors -

organophosphorus compounds, cyanides,

ions of heavy metals.

Medicinal preparations:

  Irreversible noncompetitive inhibitors of acetylcholinesterase - Phosphacol, Armin, Pyrophos.

  Hg, Bi, As are non-competitive inhibitors of harmful bacteria enzymes.

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An uncompetitive inhibitor is an inhibitor that only binds to the enzyme-substrate complex. The formation of its binding site only forms when the enzyme and the substrate have interacted amongst themselves. The uncompetitive inhibition does not work when additional substrates are trying to be involved. The enzyme-substrate-inhibitor complex does not produce any product.

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The activity displayed by enzymes is affected by a variety of factors, some of which are essential to the harmony of metabolism.

Allosteric Regulation

Enzymes regulate the rate of metabolic pathways in the cells. In a feedback inhibition, the end product of a pathway inhibits the first enzyme of that pathway. The activity of some regulatory enzymes, called allosteric enzymes, is adjusted by reversible, noncovalent binding of a specific modulator to a regulatory or allosteric site. Such modulators may be inhibitory or stimulatory and may be either the substrate itself or some other metabolite.

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Enzymes Used in Therapy

- Treatment of gastrointestinal tract diseases (digestive enzymes: pepsin, lipase, amylase, etc.)

- Treatment of purulent wounds (trypsin, chymotrypsin)

- For scars resorption (hyaluronidase)

- Treatment of viral diseases (deoxyribonuclease)

- Treatment of thrombosis and thrombophlebitis (streptodecaze, etc.)

- Treatment of some types of leukemia (asparaginase)

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Use of enzyme inhibitors

Natural proteinase inhibitors have been used in the treatment of acute pancreatitis, arthritis, allergic diseases, in which activation of proteolysis and fibrinolysis, accompanied by the formation of vasoactive kinins, is observed.

Antihypertensive drugs - Angiotensin-converting enzyme (ACE) inhibitors

Anti-inflammatory drugs (NSAIDs) are cyclooxygenase (COX) or lipooxigenase (LOG) inhibitors

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Isoenzymes

A number of enzymes exist in more than one quaternary form, differing in their relative proportions of structurally equivalent but catalytically distinct polypeptide subunits.

Investigation of isoenzymes is widely used in medical clinical practice.

CK or CPK

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Lactate dehydrogenase

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Thank you for your attention!

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Please answer the questions in chat:

1. Name the types of specificity.

2. Give an example to competitive inhibitors - medicinal preparations.

3. Point what isoforms of LDH are present in liver and in heart.

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