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Write an account on classification and nomenclature of enzymes.

Because of close interdependence, it is convenient to deal the classification and nomenclature of enzymes together. We first discuss general principles of nomenclature of enzymes. Some enzymes have been named based on the source from which they were 1st identified, e.g., papain from papaya. Name of enzyme Pepsin Trypsin Rennin Function of enzyme Hydrolyzes proteins at acidic pH Hydrolyzes proteins at mild alkaline pH Curdling of milk to start cheese making.

These are the names of enzymes ending with in, indicating that they are basically proteins. Examples of enzymes with name of substrate +ase: Lactase maltase lactoseglucose +galactose maltoseglucose.

Examples of enzymes with reaction catalyzed +ase: Glucose isomerase Lactate dehydrogenase glucosefructose lactatepyruvate

New system: The first enzyme commission recommended that there should be 2 nomenclatures for enzymes, systematic name and trivial/working name. The systematic name, formed in accordance with definite rules, showed the action of an enzyme as exactly as possible, thus identifying the enzyme precisely. The trivial name was sufficiently short for general use, but not necessarily very systematic. It was decided to retain the systematic names as the basis for classification for the following reasons: i) ii) iii) iv) The code number alone is only useful for identification of an enzyme when a copy of the enzyme list is at hand, whereas systematic name is self explanatory The systematic name stresses the type of reaction, the reaction equation does not Systematic names can be formed for new enzymes by the discoverer, by application of rules, but code numbers should not be assigned by individuals. Common names for new enzymes are frequently formed as a condensed version of the systematic names. So, systematic names are helpful in finding common names that are in accordance with the general pattern.

Scheme of classification and numbering of enzymes

Nomenclature committee for the International Union of Biochemistry and Molecular Biology (NCIUBMB) devised a system for classification of enzymes that also serves as a basis for assigning code numbers to them. EC a.b.c.d a. b. c. d. The first number shows to which of the 6 main classes the enzyme belongs The second figure indicates the subclass The third figure gives the sub-subclass Fourth figure is the serial number of the enzyme in its sub-subclass.

The tripeptide aminopeptidases have the code EC3.4.11.4. so, EC3 enzymes are hydrolases EC3.4 are hydrolases acting on peptide bonds. EC3.4.11 are those hydrolases that cleave off the amino terminal amino acid from a polypeptide EC3.4.11.4 are those that cleave off the amino terminal end from a tripeptide. Enzymes are generally classified on the basis of the types of reaction they catalyze, 6 groups of enzymes are recognized on this basis. Class 1. Oxidoreductases: To this class belongs all enzymes catalyzing oxidoreduction reactions. Substrate that is oxidized is regarded as the hydrogen donor. The systematic name is based on donor:acceptor oxidoreductase. The common name will be dehydrogenase. An alternative, reductase, can be used. Oxidase is only used in cases where O2 is the acceptor, e.g., glucose oxidase (EC1.1.3.4, systematic name, -Dglucose:oxygen 1oxidoreductase). D glucose+oxygenD glucono-1,5- lactone+hydrogen peroxide

Class 2. Transferases: They are enzymes transferring a group, like a methyl or a glycosyl group from one compound (donor) to another one (acceptor). The systematic names are formed according to the scheme donor:acceptor group transferase. The common names are normally formed according to acceptor grouptransferase or donor grouptransferase. In many cases the donor is a cofactor (coenzyme) charged with the group to be

transferred, e.g., aspartate aminotransferase (EC 2.6.1.1, systematic name, L-aspartate:2 oxoglutarate aminotransferase).

Class 3. Hydrolases: These enzymes catalyze the hydrolysis of C-O, C-N, C-C and some other bonds, including phosphoric anhydride bonds. Although the systematic name always includes hydrolases, the common name is in many cases formed by the name of the substrate with the suffix ase. It is understood that the name of the substrate with this suffix means a hydrolytic enzyme. In principle, all hydrolytic enzymes can be classified as transferases, since hydrolysis itself can be regarded as the transfer of a specific group to water as the acceptor. Yet in most cases the reaction with water as the acceptor was discovered earlier and is considered as the main physiological function of the enzyme. This is why such enzymes are classified as hydrolases rather than transferases. For example: chymosin (EC 3.4.23.4, no systematic name declared, also called rennin).

Class 4. Lyases: They are enzymes cleaving C-C, C-O, C-N and other bonds by elimination, leaving double bonds or rings, or conversely adding groups to double bonds. The systematic name is formed according to the pattern substrate group-lyase. The hyphen is an important part of the name, and to avoid confusion should not be omitted, e.g., hydro-lyase not hydrolyase. In the common names, expressions like decarboxylase, aldolase, dehydratase (in case of elimination of CO2, aldehyde or water) are used. In cases where the reverse reaction is much more important or the only one demonstrated, synthase (not synthetase) may be used in the name. For example: histidine ammonia:lyase (EC 4.3.1.3, systematic name, L-histidine ammonia-lyase; also called histidase).

L-histidine urocanate + ammonia.

Class 5. Isomerases: They catalyze geometric or structural changes within one molecule. According to the type of isomerism they may be called racemases, epimerases, cis-trans isomerases, tautomerases, mutases or cycloisomerases. The interconversion in the substrate is brought about by an intramolecular oxidoreduction (EC 5.3); since hydrogen donor and acceptor are the same molecule and no oxidized product appears, they are not classified as oxidoreductases. Even though they may contain firmly bound NAD(P)+. For example: xylose isomerase (EC 5.3.1.5, systematic name, D-xylose ketol isomerase, commonly called glucose isomerase). Class 6. Ligases: They are enzymes catalyzing the joining together of 2 molecules coupled with the hydrolysis of a diphosphate bond in ATP or a similar triphosphate. Synthetase has been used for the common names. Many authors have been confused by the use of the terms synthetase (used only for Group 6) and synthase (used throughout the list when it is desired to emphasise the synthetic nature of the reaction.) it is recommended that if the term synthetase is used by authors, it should continue to be restricted to the ligase group. For example: glutathione synthase (EC 6.3.2.3, systematic name, L-glutamyl-Lcysteine:glycine ligase (ADP forming); also called glutathione synthetase). ATP+L-glutamyl L cysteine+glycineADP + phosphate + glutathione

Give a detailed note on mechanism of action of various enzymes. Lysozyme: Lysozyme is a basic bacteriolytic enzyme that hydrolyzes peptidoglycan and is present in egg white, human tears and saliva. The egg white enzyme has 129 residues with 4 disulphide bonds. Studies with 18O-enriched water show that the C1-O bond is cleaved on the substrate between the D and E sites. This incorporates 18-O into C1. Glu-35 acts as a general acid. Asp52 forms a covalent intermediate.

Chymotrypsin: In all forms of digestion (whether of proteins, carbohydrates or fats) larger molecules are broken down into smaller molecules by the reaction with water in which a water molecule is split in two, each part joining a different product molecule. This type of reaction is called hydrolysis. Proteins are long chains of amino acids linked together by peptide bonds. When protein molecules are digested a series of hydrolysis reactions convert them into separate amino acids. The enzymatic function of chymotrypsin is to accelerate the breaking of peptide bonds that link an amino acid having non polar side chain like phenylalanine to another amino acid on the interior of polypeptide chains. a. A polypeptide substrate moves into the active site of the enzyme. The shape, size and amino acid sequence of chymotrypsins active site allow that part of the enzyme to bind a portion of a polypeptide chain that has non polar side chains like those found in phenylalanine. Once the polypeptide is in the active site, an H+ ion moves from a serine amino acid at position 195 of the enzymes amino acid sequence to the histidine amino acid at position 57. The oxygen atom in the serines hydroxyl group then forms a covalent bond to the carbon of one of the substrates peptide bonds shifting the 2 electrons from one of the double bonds up to form a lone pair. b. The plus charge formed on histidine 57 is stabilized by the negative charge on aspartate 102. When the double bond is reformed carbon and oxygen in the peptide bond, the bond between the carbon and nitrogen in the peptide bond is broken. The nitrogen containing group is stabilized by the formation of a bond to a hydrogen atom from His57. c. The portion of the polypeptide that contains the nitrogen atom from the broken peptide bond moves out of the active site. d. A water molecule moves into the active site. The oxygen atom in the water molecule loses an H+ ion to a nitrogen atom on his57. This allows oxygen atom of the water to form a bond with the carbon atom, the remaining portion of the substrate. Like in step a, one of the bonds in the double bond shifts up to form a lone pair. e. When the double bond is reformed, the bond between carbon and oxygen of serine 195 is broken. The OH group on serine 195 is restored with a transfer of an H+ ion from His57. With this step serine 195 and histidine 57 are both returned to their original forms. f. The remaining portion of the substrate moves out of the active site, leaving the active site in its original form, ready to repeat the stages a to e with another polypeptide molecule.

DNA Polymerase I: DNA Polymerase I (Pol I) is an enzyme that participates in the process of DNA replication in prokaryotes. It is composed of 928 amino acids and is an example of a processive enzyme- it can sequentially catalyze multiple polymerizations. It was initially characterized in E.coli, although it is ubiquitous in prokaryotes. In E.coli and many other bacteria, the gene which encodes Pol I is called polA. Pol I possesses 3 enzymatic activities: A 5 to 3 (forward) DNA polymerase activity, requiring a 3 primer site and a template strand. A 3 to 5 reverse exonuclease activity that mediates proofreading. A 5 to 3 forward exonuclease activity mediating nick translation during DNA repair. In the replication process, Pol I removes the RNA primer (created by Primase) from the lagging strand and fills in the necessary nucleotides of the Okazaki fragments in the 5 to 3 direction, proofreading for mistakes as it goes on. It is a template dependent enzyme, it only adds nucleotides that correctly base pair with the existing DNA strand acting as a template. Ligase then joins the various fragments together into a continuous strand of DNA. Despite its early characterization it was soon found that the Pol I was not the enzyme responsible for most DNA synthesis- DNA replication in E.coli proceeds at approximately 1000 nucleotides per second. While the rate of synthesis of Pol I averages only 20 nucleotides per second. Moreover its cellular abundance of approximately 400 molecules per cell did not correlate with the fact that there are only 2 replication forks in E. coli. Also, it is insufficiently processive to copy an entire genome, as it falls off after adding only 25-50 nucleotides.

EcoRI: EcoRI stands for Escherichia coli RI. It is an example of a specialized group of enzymes called restriction endonucleases that help bacteria to counter viral attack. These restriction enzymes can recognize specific sequences on the DNA molecule and cleave it. Thus cleaving of the DNA molecule produces restriction fragments. The sites of action of restriction enzymes are called restriction sites and are usually short palindromic sequences. EcoRI produces staggered cuts with 5 overhanging ends, but Hind III cleaves the middle of the specific sequence producing blunt ends. EcoRI cleaves DNA at GAATTC sites and serves as a paradigm for sequence specific DNA-enzyme interactions. The recently revised X-ray crystal structure of an EcoRI-DNA complex reveals that EcoRI employs novel DNA recognition motifs, a 4 alpha helix bundle and 2 extended chains which project into the major groove to contact substrate purines and pyrimidines.

Aspartate transcarbamoylase: Aspartate transcarbamoylase or ATCase catalyzes the 1st step in the pyrimidine biosynthetic pathway. In E. coli, the enzyme is a multisubunit protein complex composed of 12 subunits (300kDa in total). The composition of the subunits is C6R6, forming 2 trimers of catalytic subunits (34 kDa) and 3 dimers of regulatory subunits (17 kDa). The particular arrangement of catalytic and regulatory subunits in this enzyme affors the complex with strongly allosteric behavior with respect to its substrates. The enzyme is an archetypal example of allosteric modulation of fine control of metabolic enzyme reactions. ATCase is a highly regulated enzyme that catalyzes the 1st commited step in pyrimidine biosynthesis, the condensation of aspartate and carbamyl phosphate to form N-carbamyl-L-aspartate and inorganic phosphate. ATCase controls the rate of pyrimidine biosynthesis by altering its catalytic velocity in response to cellular levels of both purines and pyrimidines. The end product of pyrimidine pathway, CTP, induces a decrease in catalytic velocity, wheras ATP, the end product of parallel purine pathway, exerts the opposite effect, stimulating the catalytic activity.

Alcohol dehydrogenase: Alcohol dehydrogenases are a group of dehydrogenase enzymes that occur in many organisms and facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD + to NADH. In humans and many other animals they serve to break down alcohols, which could otherwise be toxic. They also participate in generation of useful aldehydes, ketones and alcohol groups during biosynthesis of various metabolites. In yeast, plants and many bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD+. In humans, the mechanism of action involves binding of the coenzyme NAD+, binding of alcohol substrate by coordination to zinc, deprotonation of His51, deprotonation of nicotinamide ribose, deprotonation of Ser48, deprotonation of alcohol, hydride transfer from alkoxide ion to NAD+ leading to NADH and a zinc bound aldehyde or ketone and finally release of the product aldehyde. ADH catalyzes the oxidation of alcohols by reducing NAD with a hydride. ADH also uses a zinc ion to electrostatically stabilize the alcohol oxygen, thus increasing the acidity of the alcohols proton. In the pathway, His 51 is activated by general base catalysis such that the histidine can then accept a proton from the NAD, which in turn draws the proton from Thr48, again demonstrating general base catalysis (although this is indirect as the substrate has not yet been involved). These proton transfers ready the threonine (which is negatively charged due to proton transfer to the NAD) for accepting a proton from alcohol of the actual substrate (cyclohexanol in the case of this particular model). This is the 1st example of true base catalysis actually involving the substrate. At the same time, since this oxidation is concerted, there is a hydride transfer to the NAD in its traditional hydride accepting region. Thus the whole sequence essentially amounts to a transfer of hydride to the NAD and the oxidation of an alcohol to an aldehyde. Key points are the orientation of the amino acid proton acceptors and donors, as well as the position of the zinc ion in relation to the substrate such that it stabilizes a negative charge on the substrate, thereby taking part in transition state stabilization.

Describe the chemistry of active site.

The active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins, except ribozymes, which is essentially RNA with catalytic activity. The active site of an enzyme is usually found in cleft or pocket that is lined by amino acid residues (nucleotides in ribozymes), that participate in recognition of the substrate. Residues that directly participate in the catalytic reaction mechanism are called active site residues. Enzymes are usually very specific as to which reactions they catalyze and substrates that are involved in these reactions. Complementary shape, charge, hydrophobic/hydrophilic characteristics of enzymes and substrates are responsible for their specificity. Enzymes can also show impressive levels of stereospecificity, regioselectivity and chemoselectivity. One of the properties of enzymes that make them important diagnostic tools is the specificity they exhibit relative to the reactions they catalyze. A few enzymes exhibit absolute specificity- 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 4 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 like amino, phosphate and methyl groups. Linkage specificity- the enzyme will act on a particular type of chemical bond regardless of the rest of the molecular structure. Stereochemical specificity- the enzyme will act on a particular steric or optical isomer. In a simple reaction, involving only one substrate, the substrate molecule binds to the active site of a particular enzyme, forming an enzyme-substrate complex. E+SES (EP) E+P

The enzyme E binds with the substrate S, forming an enzyme substrate complex ES. Following the ES complex formation, enzyme substrate interaction takes place, resulting in an enzyme-product complex (EP). In the last step, product P leaves active site of enzyme E. the released enzyme may be then recycled and combined with another substrate to form product. This way an enzyme acts on a substrate to form products. The working mechanism of an enzyme, in terms of its specificity is described by the lock and key model (Fisher) and induced fit model (Koshland). In the first model, lock represents enzyme and key is the substrate. Like a key fits exactly into its specific lock, the enzyme and substrate fit accurately into each other. As per the induced fit hypothesis, the enzyme undergoes certain structural changes after the substrate binds to the active site.

LOCK AND KEY MODEL OF ENZYME ACTION

INDUCED FIT MODEL OF ENZYME ACTION

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