Biochemistry

 

Enzyme

          Enzymes are biocatalysts that are proteinaceous (and even nucleic acids) and change the rate of a reaction. Thousands of chemical reactions take place at any given time in all of an organism's live cells. Enzymes, which are wonderful molecular machines, mediate nearly all of these processes. Enzymes are the catalysts of biological systems because they are at the heart of every metabolic reaction (biocatalysts). They catalyze hundreds of sequential reactions that degrade food molecules, store and transform chemical energy, and produce biological macromolecules from simple substrates in structured sequences. Metabolic pathways are closely coordinated through the action of regulatory enzymes, resulting in a harmonious balance among the many distinct activities required to sustain life. Enzymes catalyze an unlimited diversity of biochemical reactions because of their capacity to specifically bind a awfully wide selection of molecules. By utilizing the complete repertoire of intermolecular forces, enzymes bring substrates together in an optimal orientation, the prelude to creating and breaking chemical bonds.

They catalyze reactions by stabilizing transition states, the best energy-species in reaction pathways. By selectively stabilizing a transition state, an enzyme determines which one amongst several potential biochemical reactions actually takes place.

All enzymes are protein but all protein aren't enzymes, initially at the time of origin RNA play role as a enzyme e.g. ribozymes. Until 1980s, all enzymes were believed to be proteins. Then, Tom Cech and Sidney Altman independently discovered that certain RNA molecules may function as enzymes is also effective biocatalysts. These RNA biocatalysts have come to be called ribozymes.

An enzyme may be a protein that's synthesized during a living cell and catalyzes or hurries up a thermodynamically possible reaction so the speed of the reaction is compatible with the biochemical process essential for the upkeep of the cell. it's sometimes called as organic catalyst or biocatalyst.

Over 90% of enzymes are simple globular proteins. the rest is conjugated proteins, which have a nonprotein fraction called the prosthetic group. Many enzymes have relative molecular mass of between 10,000 and 50,000da.

The first enzyme discovered was amylase, which catalyzes the conversion of starch to maltose, in 1833 by two French chemists Payen and Persoz. However, it absolutely was not well-known until 1876 when Wilhelm Kuhne, the distinguished German biochemist, proposed the term enzyme.

Biological Importance of Enzymes:

(i) Thousands of chemical reactions are going down within the body of a living organism. All of them are mediated by enzymes.

(ii) Enzymes are specialized catalysts that operate at biological temperatures.

(iii) Enzyme mediated reactions don't require exacting treatment.

(iv) they're pH specific in order that reactions requiring different pH operate in several parts of the body.

(v) As they operate under favorable conditions, enzymes force the organisms to measure under favorable environment.

(vi) Enzymes are highly regulated. Their formation is controlled by separate genes. Activation and repression of genes allow certain enzymes to be functional or non-functional in cells.

 

 

Cofactors:

          Enzymes are composed of 1 or more polypeptide chains. However, there are variety of cases within which non-protein constituents called cofactors must be absolute to the enzyme (in addition to the substrate) for the enzyme to be catalytically active. In these instances, the exclusively protein portion of the enzyme is termed the apoenzyme. Three forms of cofactors is also identified: prosthetic groups, coenzymes, and metal ions.

Prosthetic groups are organic compounds and are distinguished from other cofactors therein they're permanently guaranteed to the apoenzyme. as an example, within the peroxisomal enzymes peroxidase and catalase, which catalyze the breakdown of peroxide to water and oxygen, heme is that the prosthetic group and may be a permanent a part of the enzyme’s site.

Coenzymes are organic compounds, but their association with the apoenzyme is transient, usually occurring only during the course of catalysis. Furthermore, the identical coenzyme molecule may function the cofactor during a number of various enzyme-catalyzed reactions. In general, coenzymes not only assist enzymes within the cleavage of the substrate but also function temporary acceptors for one in all the products of the reaction. The essential chemical components of the many coenzymes are vitamins.

For example, the coenzymes nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide -phosphate (NADP) contain the vitamin niacin; coenzyme contains pantothenic acid; flavin adenine dinucleotide (FAD) contains riboflavin (i.e., vitamin B2); thiamine pyrophosphate contains thiamine (i.e., vitamin B,), and so on.

          A number of enzymes require metal ions for his or her activity. The metal ions form coordination bonds with specific side chains at the site and at the identical time form one or more coordination bonds with the substrate. The latter assist within the polarization of the substrate bonds to be cleared by the enzyme. For example, zinc could be a cofactor for the proteolytic enzyme carboxypeptidase and forms coordination bonds with the side chains of two histidine and one aminoalkanoic acid residue at the situation. A fourth bond is made between zinc and also the a-carboxyl group of the substrate amino acids, and it's here that the cleavage of the peptide occurs.

Chemical Nature of Enzymes:

          All enzymes are globular proteins with the exception of recently discovered RNA enzymes. Some enzymes may additionally contain a non-protein group. Accordingly, there are two kinds of enzymes, simple and conjugate.

 

Simple Enzyme:

it's an enzyme which is wholly made of protein. situation is made by specific grouping of its own amino acids. Additional substance or group is absent, e.g., pepsin, trypsin, urease.

Conjugate Enzyme:

it's an enzyme which is created of two parts a protein part called apoenzyme (e.g., flavoprotein) and a nonprotein part named cofactor. the whole conjugate enzyme, consisting of an apoenzyme and a cofactor, is termed holoenzyme. site is made jointly by apoenzyme and cofactor.

Cofactor is tiny, heat stable and dialysable a part of conjugate enzyme. it should be inorganic or organic in nature. Organic cofactors are of two types, coenzymes and prosthetic groups. Coenzymes are easily separable non-protein organic cofactors. Prosthetic groups are non-protein organic cofactors firmly attached to apoenzymes, e.g., heme (= haem), biotin, pyridoxal phosphate. Heme (=haem) is iron containing prosthetic group in cytochromes, hemoglobin, myoglobin, catalase and peroxidase. The last two cause breakdown of hydrogen peroxide to water and oxygen. FMN and FAD are considered prosthetic groups by some workers while others consider them to be coenzymes. Both coenzyme and prosthetic group participate in group transfer reactions. Prosthetic group requires one apoenzyme for choosing up the group and transferring the identical. Coenzyme requires two Apo enzymes, one for selecting up the group and therefore the second for transferring the group, e.g., NAD+, NADP+, CoA.

Coenzyme has three important functions:

(a) Coenzyme is crucial for bringing the substrate connected with the enzyme,

(b) It picks up a product of the reaction, e.g., hydrogen just in case of NAD+ (nicotinamide adenine dinucleotide) or NADP+.

(c) the merchandise picked up by a coenzyme is transferred to a different reactant.

Certain workers use the term cofactor for any loosely bound non-protein group. The organic cofactor is termed coenzyme. They use the term prosthetic group similarly for both inorganic and organic group attached firmly to apoenzyme.

Most of the coenzymes are product of water-soluble vitamins, В and C, e.g., thiamine, riboflavin, nicotinamide, pyridoxine. Inorganic cofactors include ions of a range of minerals e.g., calcium, iron, copper, zinc, magnesium, manganese, potassium, nickel, molybdenum, selenium, cobalt. they typically function as activators by forming one or more coordination bonds with both the substrate and site of enzyme. Fe2+ is cofactor for catalase. Chloride ion stimulates activity of salivary amylase. Zinc is required for carboxypeptidase NAD+ and NADP+ activity.

Nomenclature of Enzymes:

All enzyme names should end in suffix ase. Exceptions are some old names, e.g., ptyalin, pepsin, trypsin. Some old names indicate the source but not the action, e.g., papain from Papaya, bromelain from Pineapple of family Bromeliaceous.

In modern system enzyme names are given after:

(i) Substrate acted upon, e.g., sucrase (after sucrose), lipase, proteinase, nuclease, peptidases, maltase

(ii) chemical change, e.g., dehydrogenase, oxidase, carboxylase, decarboxylase, etc.

          The second category of names are group names. they're often qualified by the addition of the name of substrate, e.g., succinic dehydrogenase, isocitric dehydrogenase, glutamate-pyruvate transaminase, DNA polymerase. Thus, DNA polymerase catalyzes synthesis of DNA segments through polymerization of deoxyribonucleotides. Similarly, glutamate-pyruvate transaminase transfers amino (NH2) from glutamate to pyruvate.

 

 

 

 

 

 

 

 

Classification Based upon the Reaction Catalyzed:

          Enzymes are broadly divided into six groups based on the type of reaction catalyzed.

They are:

(1) Oxidoreductases

(2) Transferases

(3) Hydrolases                                               ( O T H L I L )

(4) Lyases

(5) Isomerases and

(6) Ligases.

 

(a) Oxidoreductases:

          Enzymes which bring about oxidation and reduction reactions.

Ex. Pyruvate + NADH—lactate dehydrogenase → Lactate + NAD ⁺

Glutamic acid + NAD—glutamate dehydrogenase → α-ketoglutarate + NH₃ + NADH

(b) Transferases:

          Enzymes which catalyze transfer of groups from one substrate to another, other than hydrogen. Ex. Transaminase catalyzes transfer of amino group from amino acid to a keto acid to form a new keto acid and a new amino acid.

Ex. (α-Ketoglutarate + Alanine—alanine aminotransferase → Glutamate + Pyruvate

Aspartate + α-Ketoglutarate —aspartate aminotransferase Oxaloacetate + Glutamate

 

 

 

(c) Hydrolases:

          Those enzymes which catalyze the breakage of bonds with addition of water (hydrolysis). All the digestive enzymes are hydrolases. Ex. Pepsin, trypsin, amylase, maltase.

(d) Lyases:

          Those enzymes which catalyze the breakage of a compound into two substances by mechanism other than addition of water. The resulting product always has a double bond.

Ex. Fructose-1-6-diphosphate—ALDOLASE → Glyceraldehyde-3-phosphate + DHAP

(e) Isomerases:

          Those enzymes which catalyze the inter-conversion of optical and geometric isomers.

Ex. Glyceraldehyde-3-phosphate—ISOMERASE → Dihydroxyacetone phosphate

(f) Ligases:

          These enzymes catalyze union of two compounds. This is always an energy requiring process (active process).

Ex. Pyruvate + CO₂ + ATP—pyruvate carboxylase Oxaloacetate + ADP + Pi

 

Modes of Enzyme Action:

          There are two view points by which enzymes are supposed to bring about chemical reaction.

A. Lock and Key Hypothesis:

          It was advance by Emil Fischer in 1894. in line with this hypothesis, both enzyme and substrate molecules have specific geometrical shapes. ‘In the region of active sites, the surface configuration of the enzyme is like to permit the actual substrate molecules to be held over it. The active sites also contain special groups having —NH2, —COOH, —SH for establishing contact with the substrate molecules. The contact is specified the substrate molecules or reactants move causing the activity. it's like the system or lock and key. even as a lock are often opened by its specific key, a substrate molecule will be acted upon by a selected enzyme. This also explains the specificity of enzyme action.

After coming in grips with the situation of the enzyme, the substrate molecules or reactants form a fancy called enzyme-substrate complex. within the complexed state the molecules of the substrate undergo natural process.

The products remain attached to the enzyme for a few time so an enzyme-product complex is additionally formed. However, the products are soon released and therefore the freed enzyme is ready to bind more substrate molecules.

 

 

Enzyme + Substrate ⇋ Enzyme – Substrate Complex

Enzyme – Substrate Complex ⇋ Enzyme – Products Complex

Enzyme – Products Complex ⇋ Enzyme + Products

          Thus, we see that the chemical reactants do not cause any alteration in the composition or physiology of the enzyme. The same enzyme molecule can be used again and again. Hence, enzymes are required in very small concentrations.

B. Induced-Fit Theory:

          It is modification of lock and key hypothesis which was proposed by Koshland in 1959. According to the present theory the situation of the enzyme contains two groups, buttressing and catalytic. The buttressing group is supposed for supporting the substrate. The catalytic group is in a position to weaken the bonds of reactants by electrophilic and nucleophilic forces.

          The two groups are normally at a distance. As soon because the substrate comes connected with the buttressing group, the site of the enzyme undergoes conformational changes so on bring the catalytic group opposite the substrate bonds to be broken. Catalytic group helps in bringing about chemical change. The substrate is converted into product. the merchandise is unable to carry on the buttressing site because of change in its structure and bonds. Buttressing group reverts to its original position. the merchandise is released.

 

(A-Active site of enzymes B- Substrate Molecule C-Enzymes-Substrate complex with conformational changes so as to bring the catalytic group against the substrate bonds to be broken)

What is specific enzyme activity?

          Specific enzyme activity (usually stated simply as ‘specific activity’) is that the number of enzyme units per ml divided by the concentration of protein in mg/ml. Specific activity values are therefore quoted as units/mg or nmol/min/mg (if unit definition B is applied). Specific activity is a vital measure of enzyme purity and values for various batches of a pure enzyme should be the identical, within normal experimental error. Serial dilutions of an enzyme solution will have different enzyme activity values, but identical specific activity values because in calculating specific activity the numerator (units/ml) and denominator (mg/ml) are affected equally by sample dilution. Although specific activity is incredibly different from activity, the calculation of specific activity nonetheless depends on the activity value, and thus the stated specific activity value will be enthusiastic about the enzyme unit definition. Batches that are below the expected specific activity value may contain impurities or enzyme molecules that became denatured.

Factors affecting enzyme activity during this section we discuss why one enzyme may have different measured activity values in numerous labs. By this we mean real differences in measured activity, not apparent differences caused by the employment of various unit definitions. The conditions under which an assay is administered will influence the reported activity values.

 

 

 

 

 

 

Effects of Temperature

           

          For example, assays typically are disbursed at a temperature between 20-37o C. Generally speaking, an enzyme are going to be more active at 37o C than at 20o C. Temperature will different for various enzymes, they'll show their highest activity at its optimum temperature. because the temperature increases so does the speed of enzyme activity. An optimum activity is reached at the enzyme's optimum temperature. A continued increase in temperature leads to a pointy decrease in activity because the enzyme's site changes shape. it's now denatured.

 

 

Effects of pH

         

At very acidic and alkaline pH values the form of the enzyme is altered in order that it's now not complementary to its specific substrate. This effect is permanent and irreversible and is named denaturation. Changing the pH will affect the fees on the aminoalkanoic acid molecules. Amino acids that attracted one another may now not be. Again, the form of the enzyme, together with its site, will change. Extremes of pH also denature enzymes

 

 

 

Effect of Inhibitors

          By binding to enzymes' active sites, inhibitors reduce the compatibility of substrate and enzyme and this ends up in the inhibition of Enzyme-Substrate complexes' formation, preventing the catalysis of reactions and decreasing (at times to zero) the quantity of product produced by a reaction. Enzyme inhibitors prevent the formation of an enzyme-substrate complex and hence prevent the formation of product. Inhibition of enzymes could also be either reversible or irreversible looking on the precise effect of the inhibitor getting used.

Normal Enzyme Reaction

• In a standard reaction, a substrate binds to an enzyme (via the active site) to create an enzyme-substrate complex.

• The shape and properties of the substrate and site are complementary, leading to enzyme-substrate specificity.

• When binding occurs, the site undergoes a conformational change to optimally interact with the substrate (induced fit).

• This conformational change destabilizes chemical bonds within the substrate, lowering the energy of activation.

• As a consequence of enzyme interaction, the substrate is converted into product an accelerated rate

Competitive Inhibition: -

Competitive inhibition involves a molecule, aside from the substrate, binding to the enzyme’s situation

• The molecule (inhibitor) is structurally and chemically the same as the substrate (hence ready to bind to the active site)

• The competitive inhibitor blocks the site and thus prevents substrate binding

• As the inhibitor is in competition with the substrate, its effects may be reduced by increasing substrate concentration

Relenza (Competitive Inhibitor)

• Relenza may be a synthetic drug designed by Australian scientists to treat individuals infected with the influenza virus

• Virions are released from infected cells when the viral enzyme neuraminidase cleaves a docking protein (haemagglutinin)

• Relenza competitively binds to the neuraminidase situation and prevents the cleavage of the docking protein

• Consequently, virions aren't released from infected cells, preventing the spread of the influenza virus.

 

Noncompetitive Inhibition

·        Non-competitive inhibition involves a molecule binding to a site other than the active site (an allosteric site)

·        The binding of the inhibitor to the allosteric site causes a conformational change to the enzyme’s active site

·        As a result of this change, the active site and substrate no longer share specificity, meaning the substrate cannot bind

·        As the inhibitor is not in direct competition with the substrate, increasing substrate levels cannot mitigate the inhibitor’s effect.

Cyanide (Noncompetitive Inhibitor)

·        Cyanide is a poison which prevents ATP production via aerobic respiration, leading to eventual death

·        It binds to an allosteric site on cytochrome oxidase – a carrier molecule that forms part of the electron transport chain

·        By changing the shape of the active site, cytochrome oxidase can no longer pass electrons to the final acceptor (oxygen)

·        Consequently, the electron transport chain cannot continue to function and ATP is not produced via aerobic respiration.

Uncompetitive inhibition

           Also known as anti-competitive inhibition, takes place when an enzyme inhibitor binds only to the complex formed between the enzyme and the substrate (the E-S complex).

·        Uncompetitive inhibition typically occurs in reactions with two or more substrates or products.

·        While uncompetitive inhibition requires that an enzyme-substrate complex must be formed, non-competitive inhibition can occur with or without the substrate present.

·        Uncompetitive inhibition is distinguished from competitive inhibition by observations: uncompetitive inhibition cannot be reversed by increasing.

e.g., L-Phenyl-Alanine  Alkaline Phosphatase

Mixed inhibition

           It’s a type of enzyme inhibition in which the inhibitor may bind to the enzyme whether or not the enzyme has already bound the substrate but has a greater affinity for one state or the other. It is called "mixed" because it can be seen as a conceptual "mixture" of competitive inhibition, in which the inhibitor can only bind the enzyme if the substrate has not already bound, and uncompetitive inhibition, in which the inhibitor can only bind the enzyme if the substrate has already bound. If the ability of the inhibitor to bind the enzyme is exactly the same whether or not the enzyme has already bound the substrate, it is known as a non-competitive inhibitor. Non-competitive inhibition is sometimes thought of as a special case of mixed inhibition.

           In mixed inhibition, the inhibitor binds to an allosteric site, i.e., a site different from the active site where the substrate binds. However, not all inhibitors that bind at allosteric sites are mixed inhibitors. In gluconeogenesis, the enzyme cPEPCK (cystolic phosphoenolpyruvate carboxykinase) is responsible for converting oxaloacetate into phosphoenolpyruvic acid, or PEP, when guanosine triphosphate, GTP, is present. cPEPCK is known to be regulated by Genistein, an isoflavone that is naturally found in a number of plants. It was first proven that genistein inhibits the activity of cPEPCK.

Isoenzymes-

           Enzymes having different amino acid sequence (Different polypeptide and different gene) but carriy same reaction.

                   e.g., Hexokinase and Glucokinase

Alloenzymes-

            Enzymes produced by same gene but from different allele. Alloenzymes (or also called allozymes) are variant forms of an enzyme which differ structurally but not functionally from other allozymes coded for by different alleles at the same locus. These are opposed to isozymes, which are enzymes that perform the same function, but which are coded by genes located at different loci.

           Alloenzymes are useful in genetics and evolution but not in Biochemistry and also used for make phylogeny.

Abenzymes-

           In this case antibody acting as enzymes i.e., Catalytic antibody.

Naturally no antibody can act as enzymes.

For example, 28B4 abzyme catalyzes periodate oxidation of p-nitrotoulene methyl sulphide to sulphoxide, where electrons from the sulfur atom are transferred to the more electronegative oxygen atom.

Riboenzymes-

           Riboenzymes are catalytic RNA. Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material (like DNA) and a biological catalyst (like protein enzymes), and contributed to the RNA world hypothesis, which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems. The most common activities of natural or in vitro-evolved ribozymes are the cleavage or ligation of RNA and DNA and peptide bond formation. For example, the smallest ribozyme known (GUGGC-3') can aminoacylate a GCCU-3' sequence in the presence of Phe AMP. Within the ribosome, ribozymes function as part of the large subunit ribosomal RNA to link amino acids during protein synthesis. They also participate in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. Examples of ribozymes include the hammerhead ribozyme, the VS ribozyme, Leadzyme and the hairpin ribozyme.

Enzyme Specificity:

          Enzymes are very specific in their reaction. They either act on one particular substrate or catalyse one particular reaction.

Accordingly, enzyme specificity is of two types:

1. Reaction Specificity:

          These enzymes are specific for the type of reaction they catalyse, irrespective of the substrate on which they act. Thus, different enzymes bring about different reactions on the same substrate i.e., enzymes are specific for one particular reaction no matter which substrate it may be ex. amino acids are acted upon both by amino acid oxidase which oxidizes the amino acids to keto acids and decarboxylase that removes carbon dioxide from them.

 

 

 

 

 

 

2. Substrate Specificity:

          These enzymes are specific for the substrate upon which they act. This is further classified as follows.

(a) Absolute specificity:

          These enzymes are highly specific and act on one particular substrate only and no other substrate. Ex. Urease, catalase, aspartase.

(b) Relative specificity:

          These enzymes act on one particular bond. Ex. D-amino acid oxidase.

(c) Group specificity:

          These enzymes act on only one particular group.

i. Pepsin:

          Is a proteolytic enzyme that acts on peptide bonds contributed by aromatic amino acids like tyrosine, tryptophan and phenylalanine.

ii. Trypsin:

          Is specific for basic amino acids. Hence it cleaves peptide bonds contributed by lysine and arginine.

iii. Amino peptidase:

          Act on L-amino acids only and not on D-amino acids.

 

 

 

 

 

 

 

 

Enzymes Disorders

 

Glucose-6-phosphate dehydrogenase deficiency-

          Glucose 6 phosphate dehydrogenase deficiency could be a genetic condition that affects red blood cells, which carry oxygen from the lungs to tissues throughout the body. In affected individuals, a defect in an enzyme called glucose 6 phosphate dehydrogenase causes the premature breakdown of red blood cells. The destruction of red blood cells is termed hemolysis. If mutations within the G6PD gene reduce the quantity of glucose6phosphate dehydrogenase or alter its structure, this enzyme can not play its protective role. As a result, reactive oxygen species can build up and damage red blood cells. Factors like infections, certain medications, or ingestion of beans can increase levels of reactive oxygen species, causing red blood cells to be destroyed faster than the body can replace them. a discount within the number of red blood cells causes the signs and symptoms of haemolytic anaemia. Researchers believe that folks with a G6PD mutation could also be partially protected against malaria, an communicable disease when carried by a specific kind of mosquito. a discount within the amount of functional glucose 6 phosphate dehydrogenase seems to form it tougher for this parasite to invade red blood cells. Glucose 6 phosphate dehydrogenase deficiency occurs more frequently in areas of the planet where malaria is common.

 

 

 

 

 

 

 

Tyrosine hydroxylase

          Mutations within the tyrosine hydroxylase (TH) gene cause this impairment. This mutation is passed down through the generations as an autosomal recessive characteristic. the combination of genes for a particular trait on the chromosomes obtained from the daddy and mother determines genetic illnesses.

The TH gene encodes the instructions for creating (coding) the tyrosine hydroxylase enzyme. The organic compound tyrosine is converted to dopamine by this enzyme. Amino acids are the chemical components of proteins within the form. Dopamine could be a neurotransmitter, which could be a substance that changes, amplifies, or transmits nerve impulses from one vegetative cell to a different, allowing nerve cells to speak. Norepinephrine and epinephrine are two more neurotransmitters that are formed from dopamine (adrenaline). Dopamine is critical for the healthy functioning of the brain.

 

Phenylalanine hydroxylase

          Hydroxylase of phenylalanine this can be an autosomal recessive condition that causes intolerance to phenylalanine, a very important aminoalkanoic acid, within the diet. It affects about 1 out of each 15,000 people. Phenylalanine hydroxylase deficiency (PAH deficiency), commonly referred to as phenylketonuria (PKU), could be a hereditary disorder during which a deficient enzyme called phenylalanine hydroxylase prevents the body from correctly processing the aminoalkanoic acid phenylalanine. Mutations within the PAH gene cause PAH deficiency.

 Mr. Navnath Pawar