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CHAPTER

5

(A) Introduction

Biotechnological Applications of Enzymes

5.1 ENZYMES IN FOOD PROCESSING

One knows that, enzymes catalyze chemical changes with a speed and precision that turn a laboratory worker green with envy. One of the really exciting practical research aim today is the application of enzymes to industries such as food processing. In one sense one has been doing this at least since the start of recorded history without understanding the biochemical basis. Conversion of carbohydrates to alcohol by fermentation certainly takes advantage of yeast enzymes. In this case, however, the organism providing the enzyme remains intact and alive. The yeast cells are providing a life support system for the enzymes of fermentation. However, what is really needed is individual specific enzymes to do just what is wanted and nothing more. (B) Use of Some Specific Enzymes in Manufacture of Food Products (I) Cheese making The first step in cheese production is the precipitation (coagulation) of casein, the chief milk protein. This is done with the enzyme chymosin (rennin) obtained either from calf stomach or more recently from a microorganism. Chymosin is an aspartic acid protease which causes the coagulation of milk, a process which involves cleavage of a single peptide bond in k-casein between Phe105 and Met 106. This process releases the acidic C-terminal peptide. The high specificity of chymosin however, does not entirely depend on the recognition of residues 105 and 106 ; all the residues from 98 to 111 appear to be involved in the recognition process. The release of the C-terminal peptide is followed by Ca2+-induced aggregation of the modified micelles to form a gel. Significantly chymosin cleaves the Phe105­Met106 peptide bond in k-casein about 100 times faster than any other peptide bonds in it. Other proteolytic enzymes such as trypsin also bring about the clotting of milk, but suffer from the disadvantage of further degrading casein which leads to undesirable flavours. The flavour of foods is not dependent on proteins. The peptides and amino acids are largely responsible for sweet, sour, bitter and salty flavours. Peptides having chain lengths of 3­15 amino-acid residues and rich in hydrophobic amino acids are the major components which give bitter taste. In the search for a substitute for chymosin, enzymes that cause clotting but only limited proteolysis so as to preserve a desirable flavour are desirable. This is particulary important since now there is a reduced availability of chymosin which is obtained from calf stomachs. As said above the fungal aspartate proteases are now largely substituting for chymosin. However, these donot display a more desireable cloting/proteolysis ratio like chymosin obtained from calf stomachs. 189

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Bioorganic, Bioinorganic and Supramolecular Chemistry

The chymosin is now made by recombinant DNA technology whereby the chymosin gene has been cloned in systems like Aspergillus oryzae and is in use since 1994 for the manufacture of cheese. (II) Meat tenderizing enzymes Ordinarily meat is tough and this toughness is due to the presence of collagen, elastin and actomyosin. It is thus required to make the meat soft/tender. Moreover, the favourable flavour of meat depends largely on the concentration of peptides and amino acids in the meat. For the purpose of tenderizing, marination and flavour proteases are used. Papain is the traditional enzyme used, but others such as bromelain, trypsin, chymotrypsin and microbial proteases from Aspergillus are also used. (III) Sweeteners A very important use of enzymes is the generation of sweet syrups from cornstarch. D-glucose is not as sweet as sucrose, but D-fructose is sweeter. A mixture of D-glucose and D-fructose is sweeter than glucose alone and is used in place of sucrose in canned fruits, fruit drinks, and carbonated beverages. This mixture is prepared by use of two enzymes and these are exo-1, 4-D-glucosidase obtained from Aspergillus niger and xylose isomerase also known as glucose isomerase mainly from Streptomyces spp. Starch glucose fructose Another example of the use of an enzyme to cause an increase in sweetness is the use of -D-galactosidase in the manufacture of ice-cream. The mixture of glucose and galactose is sweeter than lactose, this enzyme can be used to catalyze an increase in sweetness of a product. (C) Immobilized Enzymes (I) The merit of using immobilized enzymes Enzymes are generally rather difficult to isolate in pure form. However, after purification, they spontaneously denature. Finally to be used, they need to be mixed with the substrate, after which it may be very difficult to get the enzymes back, even though they are perfectly reusable. A variety of techniques are solving these problems. This is done by adding cross-links to the enzyme molecule to hold the enzyme more strongly in the correct molecular pattern (I, Scheme 5.1). This prevents denaturation, extending the useful life of the enzyme. In another method the enzymes are attached to a large, insoluble support (II, Scheme 5.1). The enzymes can be added to substrate, then easily centrifuged away from the product later. It would also be possible to pack the solid supports to which enzymes were attached into a column, then pour the substrate through (III, Scheme 5.1)

-- -- -- -- -- -- -- -- -- -- -- --

-- --

--

E

E

--

E

E E

--

E

--

E

E

E E

Intermolecular crosslinkage generated by the use of reagents e.g., bifunctional reagents like 1, 5,-difluoro-2, 4-dinitrobenzene

E E

E E (II)

(I)

Support is e.g., silica glass beads

SCHEME 5.1 Continued...

Biotechnological Applications of Enzymes

191

SCHEME 5.1

(II) Immobilized enzymes--In the production of syrups, from corn starch The use of enzymes in the production of glucose syrups has been mentioned above. From 1974 onwards several million tonnes of high-fructose corn syrup is produced annually involving the use of immobilized enzymes. This advancement has largely displaced sucrose in traditional applications. Moreover, one knows that the role of fructose as a sweetening agent is much more satisfactory since it is more sweeter than glucose and also it does not crystallize readily from a concentrated solution. The two main enzyme catalyzed steps are (Scheme 5.2). The enzyme exo-1, 4--D-glucosidase (from Aspergillus niger) is immobilized on porous silica while the second enzyme is immobilized via cross linking with glutaraldehyde.

Cornstarch

exo-1, 4-a-Dglucosidase glucose

glucose

isomerase

fructose

SCHEME 5.2

(III) Role of immobilized enzymes in the synthesis of semi synthetic penicillins Several derivatives of naturally occuring penicillin are far more effective as antibiotics. These semi synthetic penicillins are made from 6-aminopenicillanic acid which is obtained from naturally occuring penicillins (Scheme 5.3). The semisynthetic penicillins have largely replaced natural penicillins and about 85% of penicillins marketed for medicinal use are semi synthetic. 6-Aminopenicillanic acid is obtained by the hydrolysis of the amide bond of the naturally occuring penicillin with the enzyme penicillin amidase, which unlike chemical hydrolysis does not open the -lactam ring.

RCONH -- CH C O

Penicillin

S N

CH3 CH3 COO

-

Penicillin amidase

+

NH3 -- CH C O N

S

CH3 CH3

-- --

6-Aminopenicillanic acid

-- --

+RCOO

-

-

COO

SCHEME 5.3

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Bioorganic, Bioinorganic and Supramolecular Chemistry

(IV) Resolution of racemic amino acid mixtures--Production of L-amino acids Synthetic methods are used for the production of huge amounts of (±)-amino acids (about 5 × 107 kg of DL-Amino acids per year). The production of chiral chemicals is necessary for biological purposes and also for use in pharmaceutical industry. Optically active compounds are needed as starting materials for asymmetric synthesis and only one of the enantiomers of a recemic mixture may have the desireable pharmacological action. These preconditions thus require the resolution of racemic mixture of amino acids by an efficient technique. The classical method of resolution employing optically active bases is both expenssive and time consuming. Recently the use of an immobilized enzyme to resolve the racemic mixture of amino acids has been developed on commercial basis. The enzyme is aminoacylase which is immobilized by binding to DEAE-Sephadex Columns (Scheme 5.4). The enzyme is specific for the hydrolysis of only L-N-acetylated amino acids. Moreover, advantage is also taken of the fact that the solubilities of free amino acids, largely differ from their N-acetylated derivatives. Following this procedure (Scheme 5.4) free L-amino acid crystallizes out from the system.

Chemical acetylation DL-Amino acid racemic mixture DL-N -Acetylamino acid

Aminoacylase D-N -Acetylamino acid +L-Amino acid

Chemical racemization D-N -Acetylamino acid

Crystallization

L-Amino acid

SCHEME 5.4

(V) Other common uses of immobilized enzymes Immobilized galactosidase generally from Aspergillus oryzae is largely used in food industry. The unwanted trisaccharide raffinose interferes with crystallization of sucrose from beet sugar syrups. Raffinose increases in concentration on storage of the beets in cold weather. An -galactosidase is used to convert the raffinose to sucrose and galactose in the syrups. Lactose malabsorption is quite common throughout the world population and this has led to a demand for lactose-free milk, which can be prepared by hydrolysing the lactose to glucose and galactose, using the immobilized enzyme -galactosidase. The carticosteriod cortisol is a useful medicine for the treatment of arthritis and it can be made from the cheap precursor 11-deoxycortisol. Two immobilized steroid enzymes 11-monooxygenase and a -dehydrogenase are used to produce prednisolone, which is a superior drug compared to cortisone (Scheme 5.5).

Biotechnological Applications of Enzymes

193

CO2OH C--O -- CH3 OH

CO2OH CO CH3 OH CH3

Steroid 11bmonooxygenase

HO CH3

O

11-Deoxycortisol

O

Cortisol

CO2OH CO CH3 OH

HO CH3

D' Dehydrogenase

O

Prednisolone

SCHEME 5.5

One future potential for such processing looks impressive. For example if a stable cellulose preparation can be researched, the cellulose of plants could be converted to glucose to be used as food. The enormous quantities of discarded cellulose in the form of paper, cotton, wood, garden debris, agricultural wastes, and food processing wastes could be converted to glucose. This glucose could then be fermented to produce ethanol. Every 12.8 lb of glucose produces a gallon of alcohol. Such ethanol could be produced at a low price enough to be considered as a supplement to or replacement for petroleum fuels in automobiles. This process would help solve both an energy problem and a garbage disposal problem. Immobilized enzymes are made by the attachment of an enzyme to an insoluble support which allows its reuse and continuous use and thus eliminating the tedious recovery process. Immobilization stabilizes the enzyme, moreover two or more enzymes catalyzing a series of reactions may be placed in close proximity to one another. Adsorption, covalent linkage, cross linking, matrix entrapment or encapsulation are different methods for making immobilized enzymes. Production of glucose syrups from starch by the use of immobilized enzymes is one of the most important processes of food industry.

5.2 CLINICAL USES OF ENZYMES

(A) Introduction It was first discovered in 1954 that the activity of enzyme aspartate aminotransferase in serum increased shortly after myocardial infraction [myocardial infraction is defined as necrosis of myocardium (heat muscle) due to cessation of blood flow i.e., a process of formation of an area of dead heart muscle]. This observation has led to widen the scope of measurement of enzyme activity in such body fluids as plasma or serum and has become a valueable tool in medical

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Bioorganic, Bioinorganic and Supramolecular Chemistry

diagnosis (clinical enzymology). Certain enzymes that function in the plasma, such as the enzymes involved in blood clotting, are continually secreted into the blood by the liver. Most other enzymes, however, are normally present in plasma in very low concentrations. They are derived from the routine destruction of erythrocytes, leukocytes, and other cells. When cells die, their soluble enzymes leak out of the cells and enter the bloodstream. However, all cells donot contain the same complement of enzymes, those that are specific to a particular organ can be important in aiding diagnosis. Therefore, an abnormally high level of a particular enzyme in the blood often indicates specific tissue damage, as in hepatitis and myocardial infarction (myo, muscle ; cardi, heart ; an infarct is an area of dead tissue). For example, elevated blood levels of creatine kinase (CK) and glutamic-oxaloacetic transaminase (GOT) accompany some forms of severe heart disease. A blood analysis that show high levels of CK may indicate that the heart muscle has suffered serious damage. (B) Clinical enzymology of heart disease Several enzymes are present in the heart muscle (myocardium). When the blood flow to the myocardium is interrupted, myocardial necrosis (heart attack) occurs. The enzymes present in the myocardium leak into the circulating blood. Measurement of these enzymes is useful in detecting myocardial infarction. The following enzymes are present in myocardium : creatine kinase (CK), lactate dehydrogenase (LDH) and Glutamic-oxaloacetic transaminase (GOT). Creatine kinase is the earliest to be detectable rising 4-6 hours after the chest pain, reaching a peak at 24-36 hour and then rapidly declining (Scheme 5.6). Creatine kinase is also present in skeletal muscle and brain. In skeletal muscle this enzyme has almost eight times the concentration on a gram wet weight basis compared to that of cardiac muscle. Generally an increase in CK activity in serum is mostly associated with damage to the cardiac or skeletal muscle and less frequently to brain damage. Moreover since some of these enzymes are also present in other organs such as liver, lungs and red blood cells, hence they are not specific to the heart. To further refine the detection of infarction various sub-types of these enzymes have been identified. For example CK has three isoforms i.e., BB, MB and MM. BB isoform is present mostly in the brain, MM mostly in the skeletal muscle and MB mostly in the heart. Measurement of MB isoform is, therefore, most useful in making the diagnosis of infarction.

Extent of enzyme increase (multiple of normal)

5X 4X 3X 2X Creatine kinase Glutamic oxaloacetic transaminase Lactic dehydrogenase

1 (Normal) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Days after episode Chest pain

SCHEME 5.6

Biotechnological Applications of Enzymes

195

Modern medical practices have automated and computerized the assay procedures for most of these serum enzymes. It is important to note that the precise patterns of enzyme changes in certain tissue diseases are characteristic. For example, in a myocardial infarction, the GOT/GPT ratio is usually high ; the reverse is true in liver disease. (C) Clinical enzymology of liver disease The aminotransferases (formerly called transaminases), alkaline phosphatase and GGT are present in liver cells. With liver cell injury or death, these enzymes leak into the blood stream. Measurement of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) is particularly helpful in detecting liver disease. (D) Clinical enzymology of cancer Certain enzymes such as L-aasparaginase have been found to be useful in treating cancer. L-aasparagine is a non-essential amino acid that is required by cancer cells for their growth. L-aasparaginase occurs in plants, animals and bacteria. Most common mammals lack this enzyme. L-aasparaginase is mostly derived from the bacterium Escherichia coli. Laasparaginase by lowering the concentration of asparagine retards the growth of cancer cells. It has proven particularly useful in treating lymphoblastic leukemia and certain forms of lymphomas. (E) Clinical enzymology of pancreatitis -Amylase is largely present in the slivary glands and in the pancreas. This enzyme is an endoamylase which brings about the hydrolysis of the 1, 4- linkage in amylose and amylopectin (Scheme 5.7). Low emylase activity is detected in the serum and urine of normal subjects. In the case of acute pancreatitis the amylase activity increases 20-30 times the normal levels. Significantly, acute pancreatitis would be otherwise diffcult to diagnose in the absence of enzyme tests since the patient normally complains of intense upper abdominal (the area where pancreas are located) pain which could be due to several other ailments. Infact both -amylase and lipase are elevated in acute pancreatitis.

b-1,4-glycoside bond

CH2OH O HO O

1

CH2OH O

4

OH O O HO CH2OH Cellulose HO O O

HO O

CH2 O HO O

HO

HO

a-1,4-glycoside bond

a-amylose

Humans do not have enzymes which can hydrolyze b-glycoside linkage and thereby utilize the component glucose

SCHEME 5.7

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Bioorganic, Bioinorganic and Supramolecular Chemistry

5.3 MUTATIONS AND GENETIC DISEASES-FAILURE TO SYNTHESIZE A PARTICULAR ENZYME

(A) Enzyme deficiencies and associated diseases-phenylketonuria One knows that DNA directs the synthesis of proteins and the sequence of bases in the DNA is critical and specific for the proper sequence of amino acids in proteins. Occassionally, however, the base sequence in DNA may get modified by exposure to heat, radiation or some chemicals leading to mutation. [Mutation is altering of genetic information in such a fashion that the message for sequence of amino acids of a specific protein is altered.] Thus bases in DNA can be altered or lost, the phosphodiester bonds in the backbone can be broken and strands can become covalently cross-linked. Thus e.g., some chemicals including acids and oxidising agents can modify DNA by alkylation, melhylation or deamination. DNA is also susceptible to spontaneous loss of heterocyclic bases (depurination or depyrimidization). [In replication of DNA alone, each time a human cell divides, a copy is made of 4 billion bases to generate a new strand of DNA. Probably there are 2000 errors each times replication occurs. Several of these errors are, however, not important, but many may lead to genetic diseases. Those diseases result from the inability to produce one of the enzymes of a metabolic pathway in an active form (a situation called genetic deficiency)]. In most genetic diseases, the resulting defective genes lead to the failure to synthesize a particular enzyme. One has already learnt that elevated enzyme activities in serum and urine form the basis of diagonsis of associated diseases. Often, on the other hand some subjects may suffer from enzyme deficiencies as a result of inborn errors in metabolism and about 400 such inborn errors (genetic diseases) in melabolism are now recognized. Phenylketonuria is one such inborn disorder (1 in 12,000 births in UK) and in most of these situations an enzyme deficiency has been recognized. In fact in many cases the presence of a metabolite is used for the diagnosis of the desease e.g., phenylketonuria is detected by estimating the metabolite phenylpyruvate in urine or phenylalanine in blood (Scheme 5.8). In phenylketonuria there is lack of the enzyme phenylalanine hydroxylase which is needed to convert phenylalanine to tyrosine (Scheme 5.8) which is the precursor of the neurotrans-mitters dopamine and norepinephrine as well as the skin pigment melanin. Phenylalanine is an essential amino acid which is formed regularly in a normal subject due to protein turnover and dietary in take. In a person suffering from phenylketonuria (a phenylketonuric subject) there is a high build up of phenylalanine or its metabolites. Phenylketonuria is associated with mental retardation and the desease can be controlled by

CH2CHCOOH NH2

phenylalanine transamination Phenylalanine hydroxylase

CH2CHCOOH NH2 HO tyrosine

O CH2--C--COOH

Tyrosine also serves as a precursor for the melanins, the pigments which color the skin, hair and eyes. This leads to albinism.

phenylpyruvate

SCHEME 5.8

Biotechnological Applications of Enzymes

197

using a diet with reduced phenylalanine content. On reducing the protein content in the normal diet would lead to deficiency in other essential amino acids. This has led to semisynthetic diets for phenylketonuric persons which contain protein hydrolysate from which only the phenylalanine contant is reduced. Thus preventing mental retardation, in a phenylketonuric person. O A recent problem of significance is the use of the artificial H C sweetener arpartame [aspartame, a dipeptide was discovered in COOH N 1969 is about 200 times sweeter than sugar and is made from NH2 H OCH 3 amino acids phenylalanine and aspartate] which has significantly replaced other sweeteners (Scheme 5.9). Aspartame is convertad H O into L-phenylalanine, L-aspartic acid and methanol in the intestine. This has led to the importance of early detection of phenylketonuria and infact in some countries, screening is done aspartame right at birth.

SCHEME 5.9

(B) SOME EXAMPLES OF DAMAGE TO DNA (GENETIC-DISEASES) AND THEIR ENZYMATIC REPAIR

(I) Lesions in DNA-pyrimidine dimers formed by ultraviolet light and their repair-enzyme DNA photolyase On exposure to UV light two adjacent thymines of a DNA strand can become covalently linked to give a thymine dimer. It is an example of photodimerization. DNA replication cannot take place in the presence of such a dimer (Scheme 5.10) since a dimer distorts a template strand (I, Scheme 5.10). Thus the removal of the pyrimidine dimers is necessary for survival. The repair process involves an enzyme known as DNA photolyase. This enzyme recognizes and binds to the thymine dimer. In the presence of visible light the enzyme catalyzes the cleavage

O O O O

--

--

CH3 CH3

CH3 H3C

--

--

H--N

N--H

H--N

N--H

--

--

--

S P P

A single strand of DNA with two adjacent pyrimidines (Thymines)

P

P

P

Formation of a thymine dimer

-- -- --

--

S

O

N

N

O

UV

O

N

N

O

P

3' 5'

(I)

5' 3'

SCHEME 5.10

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Bioorganic, Bioinorganic and Supramolecular Chemistry

of the dimer and thus restores normal base pairing and thus DNA is repaired. The enzyme (photolyase) then dissociates from the repaired DNA leading to the normal A/T base pair reforming. The joined thymines called dimers prevent the replication of DNA. Several enzyme systems in normal cells recognize these dimers and either reverse the dimerization or cut out and replace the altered section of DNA. People suffering from (XP = Xeroderma pigmentosum­development of dry skin, skin and eye cancers) lack any one of the nine enzymes necessary for repairing this demage to the DNA. (II) Mutations caused by changes in the dase sequence of DNA One may consider a situation when one base pair is substituted by another. Some of the hydrogen atoms on each of the four bases may change their location to give a tautomer e.g., an amino group (--NH2) can tautomerise to an imino form (= NH) while a keto group (C = O) tautomerizes to an enol (= C--OH). These tautomers though transient can lead to unusual base pairs which can fit into a double helix. Thus the imino tautomer of adenine can pair with cytosine (Scheme 5.12). This unusual pairing of A*--C (asterisk denotes the imino tautomer) then leads to C DNA-bases One may recall the structures of the four DNA bases and the Watson and Crick specificity of their pairing adenine (A) must pair with thymine (T) and guanine (G) with cytosine (C) i.e., A--T and G--C (Scheme 5.11)

H C

1 6 3

N

5

C

N

7 8 CH 9

N

H C

3 4 1

5

CH

HC 2

4C

N

Purine

N H

HC 2

6 CH

N

Pyrimidine

NH2 N C

1 6 3 5

O C N

7 8 C--H 9

O C N

7 8 C--H 9

NH2

5

HN

C

1 6 3

5

H--N

C

3 4

C--CH3 O

N

C

3 4

5

C--H

H--C 2

4C

N

Adenine (A)

N H

H2N--C 2

4C

N

Guanine (A)

N H

O--C 2

N H

1 6 C--H

C2

N

1 6 C--H

H

Cytosine (C)

Thymine (T)

SCHEME 5.11

Biotechnological Applications of Enzymes

H H C N C 1¢ C O

Cytosine

199

H N C N H N1 C H H N C

6

C

H The rare tautomer of adenine pairs with cytosine instead of thymine. This tautomer is formed by the shift of a proton from the 6-amino group to N-1.

C C

N C--H N C 1¢

N

Rare tautomer of adenine

SCHEME 5.12

getting incorporated into a growing strand where istead normally it should have been T and this leads to mutation. Hydrolytic deamination of cytosine gives uracil which pair with adenine rather than guanine (Scheme 5.13)--another situation which damages DNA.

H N C HC HC N N C O

Hydrolytic deamination H 2O NH3

H O C HC HC N

Uracil

NH C O

Cytosine

SCHEME 5.13

Most of these defects in DNA are repaired by general excision-repair pathway. In the first step of the repair pathway an enzyme­endonuclease recognizes the distorted, demaged DNA and both ends of the lesion are excised. This enzymatic cleavage releases an oligonucleotide with about 12 residues leading to a gap. This gap is filled by a DNA polymerase and the nick is sealed by DNA ligase (Scheme 5.14).

(C) ROLE OF ENZYMES IN DRUG DESIGN

(I) Lowering of Cholesterol Levels One has learnt the clinical situations where the enzyme overactivity is used to detect some diseases like heart disease. A deficiency of a particular enzyme may be the cause of a genetically inherited disease like e.g., phenylketonuria. It is now well established that many drugs function through their inhibitory effect on a critical enzyme in the cells of the invading organism. Another genetically inherited disease is hypercholesterolaemia in which there is a high circulating level of cholesterol/cholesterol esters in the blood (generally 4-6 times the normal circulating levels of cholestrol). The disease infact involves a protein rather than an enzyme defect. The disease leads to the deposition of arterial plaques with a consequent symptoms of cornonary

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Bioorganic, Bioinorganic and Supramolecular Chemistry

Site of damage--a region e.g., containing a thymine dimer

3' Damaged DNA 5'

5'

3' Excision of a 12 nucleotide fragment by endonuclease nicks the DNA backbone on both sides of the damage (see arrows) 5'

3'

5' A helicase removes the damaged DNA, leaving a gap. 3'

3'

5'

5' DNA synthesis by DNA polymerase fills the gap 3'

3'

5'

5' Joining by DNA ligase.

3'

3' Normal DNA 5'

5'

3'

SCHEME 5.14

disease in childhood or adolescence. The disease is due to deficiency in the low density lipoprotein receptors which are involved in the uptake of cholesterol/cholesterol esters by the tissues leading to an increase of levels of cholesterol/cholesterol esters. These high levels of circulating cholesterol/cholesterol esters can be brought down and thus also the tendency for the formation of arterial plaques by giving a drug­a competitive inhibitor of hydroxymethyl-glutaryl CoA reductase (HMG CoA reductases). HMG CoA reductase catalyses the rate-limiting step in the formation of cholesterol (Scheme 5.15). Three competitive inhibitors of HMG CoA reductase

Biotechnological Applications of Enzymes

HMG CoA + 2NADPH + 2H

+

201

mevalonate + 2NADP

+

CH3 HO

C OH

O O

-

H O C O HO O

O O O C

CH3

HO

O SCoA

CH3

Mevinolin Compactin R=H

R

Monacol K R = CH3

HMG CoA

SCHEME 5.15

(Scheme 5.15) have been researched which inhibit the enzyme. All three are fungal products and have a structural resemblance to HMG CoA. Mevalonate is a precursor not only of steroids but also of a number of other important cell constituents. Sterol biosynthesis appears to be selectively inhibited at low concentrations of the enzyme (also see Scheme 4.46). (II) Role of sulfa drugs One of the best understood antimetabolites is the synthetic sulfa drug sulfanilamide which interferes with the normal metabolism of p-amino-benzoic acid, due to their close structural similarity (Scheme 5.16). The following points may be considered:

CO2H H N N H CH2 H CH2--N--

9 10

H2N HN

2 3

N

1 4

8 7 5 6

CH2 H --C--N--CH--CO2H O

O O H2N--

--C--OH

H2N--

--SO2NH2

p-aminobenzoic acid

p-aminosulfonamide, a sulfa drug

SCHEME 5.16

· p-Aminobenzoic acid is vital for the growth of many pathogenic bacteria. · The vitamin folic acid serves as a coenzyme for several important biochemical processes. Folic acid is obtained by a human from its diets and from the bacteria in the digestive tracts.

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Bioorganic, Bioinorganic and Supramolecular Chemistry

· Bacteria can synthesize folic acid from available p-aminobanzoic acid, however, in the presence of structurally similar drug sulfanilamide, the bacterial enzyme easily incorporates instead the drug to produce a false nonfunctional folic acid. · This false folic acid cannot act as a proper coenzyme and it also acts as a competitive inhibitor for the enzyme. · Thus the bacteria are unable to biosynthesize vital compounds like certain amino acids and nucleotides for their survival and they die (also see Scheme 5.24). (III) Penicillin antibiotics Lastly mention may be made of several natually occuring penicillins which have structural similarity. All have the empirical formula C 9 H 11 O 4SN 2R, with a four membered ring fused to a five membered ring. A variety of structural variations in R groups are obtained by adding an appropriate organic compound to the culture medium. An antibiotic is a compound which is produced by one microorganism (bacterium, mold yeast) and is toxic to another.

O R--C N H O

a penicillin Cysteine

H C C

H C N

S

CH3 CH3

COOH

valine

A penicillin (Scheme 5.17) is the most widely used SCHEME 5.17 antibiotic in the world and functions by inhibiting an enzyme (transpeptidase) which catalyzes the last step in bacterial cell wall biosynthesis leading to death of bacteria. (IV) Cancer drugs and biosynthesis of nucleotides Nucleotides are involved in the transfer of hereditary information and numerous other reactions and are thus crucial to life and all nucleotides can be made by the body and a dietary source is not needed. Again, each nucleotide has its own biosynthetic pathway. Deoxythymidine

O C HN O

-

O C CH HN O P O

-

C--CH3 N C--H + DHF

O O CH2 H H OH

dUMP

C

N

CH

O

P O

O H H

Thymidylate Methylene synthase + O THF

O O CH2 H H OH

dTMP

C

O H H

H

H

SCHEME 5.18

Biotechnological Applications of Enzymes

203

monophosphate (dTMP) is formed from deoxyuridine monophosphate (dUMP) by the donation of a methyl group by a coenzyme derived from folic acid (methylene THF) one has already seen (Scheme 1.26) that uracil has the same shape and size as the drug 5-fluorouracil. This is the basis of discovery of 5-fluorocil as a cancer drug. Cancer cells divide more rapidly than most normal cells. Cell division requires DNA replication, which in turn requires the deoxynucleotides. Inhibition of dTMP synthesis kills rapidly dividing cells. The drug 5-fluorouracil is metabolized to an inhibitor of thymidylate synthase, a key enzyme in the synthesis of deoxytymidylate. This nucleotide is needed for the synthesis of DNA, which must be synthesized before cell division can occur. If there is little or no DNA synthesis, there will be little or no cell division (Scheme 5.18). Of the side effects of cancer chemotherapy can be traced to the loss of normal rapidly dividing cells. Hair cells die, and cells lining the gastrointestinal tract are affected as are the cells that divide to form blood cells. Hair loss, gastrointestinal problems, and suppressed immunity are all side effects of chemotherapy (also see Problem 2.13).

5.4 RECOMBINANT DNA TECHNOLOGY (GENETIC ENGINEERING OR CLONING)

(A) Introduction-Understanding recombinant DNA technology DNA, the genetic material is a long, unbranched polymer. The gene may be regarded as a segment of the DNA molecule which directs the synthesis of all the protein molecules made by the cell. DNA molecules are among the longest molecules known. The DNA found in one human cell has about 5.5 billion nucleotide base pairs which probably make up 1 million genes. Recombinant DNA technology is the transplantation of genes from one organism into another. It depends first on having enzymes that can cleave DNA chains into specific fragments which can be manipulated. A new piece of DNA is then inserted into a gap created by cleavage and resealing the chains (ligation of DNA fragments with DNA ligases) and its introduction into host cells. When the recombination is successful, the gene which is transplanted will express (synthesize) its normal protein product thus bacteria which receive human genes can be induced to express human proteins of value to treat a particular disease. In short recombinant DNA technology is based on nucleic acid enzymology, which involves alteration of DNA of some organism with a goal of having that organism produce a desired protein. Recombinant DNA technology has produced useful proteins for human therapy. One example is the use of insulin. The insulin obtained from hogs has a similarity in amino acid composition to human insulin, however, insulin from this source often causes allergic response. Human insulin produced by bacteria via recombinant DNA technology is on the market for use by diabetic patients. The recombinant DNA technology can thus be studied via the study of the following overall aspects : · The structural outline and properties of DNA · DNA replication--use of DNA polymerases · Cleavage of DNA duplex at specific sequences--use of enzymes--restriction endonucleases

204

Bioorganic, Bioinorganic and Supramolecular Chemistry

· Sealing of gap in DNA--use of DNA ligases. · Introduction of modified DNA into host cells and the study of replication and expression of recombinant DNA in host cells. (B) Structure of DNA This may be studied by looking to the following points : · The molecular mass of a DNA molecule is very high as several billion, on the other hand RNA molecules are much smaller­generally falling in the range 20,000 to 40,000. · DNA is very long thread like macromolecule which is composed of a large number of deoxyribonucleotides and each of these is composed of a nitrogen base, a sugar and a phosphate group. The sugar is deoxyribose (see Scheme 1.27) while four different nitrogen bases (heterocyclic amines) are found in DNA. Two of these bases adenine (A) and guanine (G) are derivatives of purine, the other two thymine (T) and cytosine (C) are derivatives of pyrimidine (see Schemes 1.26 and 1.27). · The sequence of nucleotide bases of DNA molecules carry the genetic information (Majority of plant and animal genes occur in pieces spread out along the DNA) while their sugar and phosphate groups plays the structural role. [using DNA as a pattern or template, the genetic information is transferred to RNA. The RNA enters the cytoplasm and controls the order in which the amino acids are assembled into new protein] · The structures of four deoxynucleoside monophosphates (or nucleotides) are in (Scheme 5.19) and represent deoxyadenosine monophosphate (dAMP), deoxythymidine monophosphate (dTMP), deoxyguanosine monophosphate (dGMP), and deoxycytidine monophosphate (dCMP). These are linked in the polymer by an ester bond between the 5-phosphate of the nucleotide and the 3-hydroxyl of the sugar of the next as shown (Scheme 1.27). One end of this linear polynucleotide is said to be 5 (as no residue is attached to its 5 carbon) and the other is said to be 3 (since no residue is attached to its 3-carbon).

O

-

­

O O N N O H

2'

-

O

P O

5' CH2

NH2 N N

-

O

P O CH2

O H 3C

O NH N H H O

O

H

H OH

H

H

H OH

H

H

2'-Deoxyadenosine 5'-monophosphate (Deoxyadenylate, dAMP) (I)

2'-Deoxythymidine 5'-monophosphate (Thymidylate, dTMP) (II)

SCHEME 5.19 Continued...

Biotechnological Applications of Enzymes

205

O

-

O

-

-

O

P O CH2

O N N O H H

O NH N NH2

-

O

P O CH2

O

NH2 N O H H N O

H

H OH

H

H

H OH

H

2'-Deoxyguanosine 5'-monophosphate (Deoxyguanylate, dGMP) (III)

2'-Deoxycytidine 5'-monophosphate (Deoxycytidylate, dCMP) (IV)

SCHEME 5.19

· The sugar units and phosphate groups are same in all the nucleotides thus the structural abbreviations repersent only the sequence of the nitrogen bases. By convention one starts at the left working in the 5 -- 3 direction. Thus a tetranucloetide can be referred to as A --G -- T --C when it is clear that the reference is to DNA. On may

5 3

often write these abbreviations for the base sequence in DNA by putting a lower case d in front of the base sequence i.e., dA -- G -- T -- C . This shows that all the sugar

5 3

units of the sugar-phosphodiester backbone of the molecule are deoxyribose in DNA (since the phosphate ester bridges holding the nucleotides together each contain two phosphate ester linkages, these bridges are called phosphodiesters). · These polymeric molecules, consist of two strands of polynucleotide and each base of one strands forms hydrogen bonds with a base of the opposite strand to give a base pair (I, Scheme 5.20), commonly between the lactam and amino tautomers of the bases. · Because A in one strand pairs with T in the other strand while G pairs with C, the strands are complementary. In any DNA molecule, A = T and G = C. · The strands of DNA run in opposite directions i.e., these are antiparallel. Each end of double-stranded DNA is made up of the 5 end of one strand and the 3 end of another. This base pairing, thus enables two complementary strands of DNA to form a duplex. Whose shorthand structure may be represented as (I, Scheme 5.20). · The duplex is in fact a right-handed double helix (II) (Watson and Crick, the Noble prize 1962) where the two helical polynucleotide chains wrap around around each other (two stranded helical structure around a common axis). Thus the DNA molecule can be imagined as a "ladder" which has been twisted into a helix.

206

Bioorganic, Bioinorganic and Supramolecular Chemistry

SCHEME 5.20

· The purine and pyrimidine bases are on the inside of the helix while the sugarphosphate units on the nucleotide are on the outside of the DNA molecule. The planes of the bases are perpendicular to the helix axis, while the plane of the sugars are almost at right angles to those of the bases. · The precise sequence of bases carries the genetic information while the Watson-Crick pairing rules for bases that adenine (A) must pair with thymine (T) ; and guanine (G) with Cytosine (C), (specificity for the pairing of bases) reflect on the double helical structure regarding steric and hydrogen bonding factors. There is just the right amount of space in the center of the helix for one purine and one pyrimidine to fit across from each other (III Scheme 5.20). In contrast the room is insufficient for two purines, there is more than enough space for two pyrimidines and in that case these would be too far apart to form effective hydrogen bonding. Thus out of the base pair in a DNA helix one must always be a purine and other pyrimidine (steric factors). The base pairing in further restricted by hydrogen bonding requirements. When thymine is across from adenine, the hydrogen bond can form at two places, however, if thymine were across from guanine only one bond would be

Biotechnological Applications of Enzymes

207

possible. Thus adenine and thymine always pair. In the case of guanine and cytosine, three hydrogen bond can form, adenine would be unable to hydrogen bond to cytosine under the same conditions. Guanine and cytosine are thus also uniquely suited to pair. · The DNA molecules from many sources are circular. (C) DNA replication by DNA polymerases that take instructions from temptates The DNA can be replicated by an enzyme DNA polymerase and replication proceeds exclusively in the 5 ---- 3 direction. DNA polymerase catalyses the step by step addition of deoxyribonucleotide units to a DNA chain (Scheme 5.21). The synthesis of DNA uses as starting materials the nucleoside 5 triphosphates of the bases found in DNA (the abbreviation dNTP represents any deoxyribonucleoside triphosphate and PPi denotes the pyrophosphate group). The specific nucleotide unit to be added under the DNA polymerase catalysis is determined by Watson­Crick base pairing to the template strand, adenine (A) pairs with thymine (T) and guanine (G) pairs with cytosine (C) as shown (Scheme 5.22).

Base O H H H

O

O

O

(DNA)n residues + dNTP

(DNA)n+1 + PPi

-

O--P--O--P--O--P-- OCH2 O

-

O

-

O

-

H

H HO

dNTP

SCHEME 5.21

DNA polymerase catalyzes the formation of a phosphodiester bond (see arows) only if the base on the incoming nucleotide is complementary to the base on the template strand. Thus e.g., the base of dNTP must be thymine (T) so as to match with adenine (A) on template strand.

Chain-elongation reaction catalyzed by DNA polymerase SCHEME 5.22

Thus for DNA replication using DNA polymerase as a catalyst one must have a template strand of DNA, (template­a polymer molecule whose sequence is used for charting a sequence for another molecule. DNA serves as a template for DNA synthesis during replication) a DNA

208

Bioorganic, Bioinorganic and Supramolecular Chemistry

3' DNA

H H

New strand

5' DNA

O H O CH2 O O H P O H O CH2 O

5' DNA

N O N N N (G) H H N N H CH3 O H N H N N N (A) H N N H N N O (C)

H

H

H

O CH2 H

3'

O H

2'

O

-

H

H

H

O O O O H

H

H

H

O --P--O--P--O--P--O O O O

5' CH2

O (T)

Hydrogen bonds have formed

O H H H H OH H

N

Template strand

dNTP

3' DNA

H H

New strand

5' DNA

O H O CH2 O O H P O H O CH2 O

5' DNA

N O H H N N N H H N O N

H H

H

O CH2 H

3'

N N H

2'

O

O

-

H

H O H N N H H N N H H N N

CH3

H

O O-- P O

5' CH2

H O

H

H

N O

O

H

H OH

Template strand

H

SCHEME 5.23

Biotechnological Applications of Enzymes

209

or RNA primer which is annealed to the tamplate and which has a 3 hydroxyl group on the deoxyribose and an appropriate dNTP as a source of monomers (Scheme 5.22). The shorthand representation shown (Scheme 5.22) for elongation of the DNA chain is elaborated in (Scheme 5.23). The incoming dNTP forms a base pair with the residue on the tamplate strand and after this correct base pair is formed, the 3­OH group of the primer carries out a nucleophilic attack on the -phosphorus atom (innermost phosphorus atom) of the incoming dNTP. This leads to the addition of a nucleoside monophosphate residue with displacement of pyrophosphate. The subsequent hydrolysis of the pyrophosphate (PPi not shown) makes the polymerization reaction essentially irreverssible. As true in the case of other nucleotide reactions which release pyrophosphate, the presence of Mg2+ is a must, Mg2+ complexes with the and -phosphate groups to make nucleoophilic attack feasible. Thus one may simplify the information (Scheme 5.23 to Scheme 5.24). In summary the DNA is replicated by DNA polymerase by taking instruction (presence of a suitable base) from template. The dNTP forms a phosphodiester bond provided it has a complimentary base to the base on the template strand. The shorthand representation is in (Scheme 5.22) which is elaborated for understanding in (Scheme 5.23) and may be represented as in (Scheme 5.24). The DNA polymerase is a template­directed enzyme. The enzyme takes instructions from the template leading to a synthesis of a feature with a base sequence complimentary to that present in the template. (D) Restriction enzymes split DNA in to specific fragments Restriction enzymes (restriction endonucleases) recognise specific base sequences in double helical DNA and bring out cleavage of both strands of the duplex in regions of defined sequence. One of their important uses is in recombinant DNA technology. Restriction enzymes cleave foreign DNA molecules. The term restriction endonuclease comes from the observation that certain bacteria can block virus infections by specifically destroying the incoming viral DNA. Such bacteria are known as restricting hosts, since they restrict the expression of foreign DNA. Restricting hosts synthesize nucleases which digest foreign DNA. The cell's own DNA is not degraded by restriction enzymes since it is methylated at critical base sites at the sites recognized by endonucleases. In fact many bacteria have at least one highly specific restriction endonuclease as well as a restriction methylase of identical specificity (this protects the host DNA by methylating critical bases). Many restriction enzymes recognize specific sequences of four to eight base pairs and bring about the hydrolysis of a phosphodiester bond in each strand in this region and nearly in all cases the recognition sites have a two fold axis of symmetry (C2 axis) i.e., the 5 -- 3 sequence of residues is the same in both the strands of the DNA molecule. Thus the paired sequences "read" the same in either direction (one says that the recognized sequence is polindromic, these sequences are termed palindromes­palindromes in English include BIB, DEED, RADAR and MADAMI' MADAM) and the cleavage sites are symmetrically positioned. EcoRI was one of the first restriction enzymes to be discovered. It is present in many strains of Escherichia coli this enzyme has a palindromic recognition sequence of six base pairs (the 5 3 sequence is GAATTC on each strand). This endonuclease catalyzes the hydrolysis of the phosphodiesters which link G to A in each strand and thus DNA is cleaved (Scheme 5.25).

210

Bioorganic, Bioinorganic and Supramolecular Chemistry

O

Primer strand

O

O O O H HO H H A H

O--P--O--P--O--P G O C--D N A t e m p l a t e s t r T-- a n d O O O H 2C H

O H 2C H H HO

H H

H

elongation of DNA chain to occur here

appropriate dNTP Polymerase

Primer strand

Primer strand

O H 2C H H O O--P O H 2C H H HO O H H A H O O H H G H D C-- N A t e m p l a t e T-- s t r a n d

O H 2C H O O H H O O O O H HO H H A H O H H G H D C--N A t e m p l a t e T-- s t r a n d

O --P--O--P--O--P O O O H 2C H

DNA polymerase brings about the synthesis of DNA by adding one nucleotide, unit at a time to the 3 end. The nucleotide substrate is a suitable deoxyribonucleoside 5 -triphosphate (dNTP). The specificity of the dNTP whether dATP, dGTP, dTTP and dCTP is determined by Watson-Crick base pairing to the template strand ; adenine (A) pairs with thymine (T) and guinine (G) pairs with cytosine (C).

SCHEME 5.24

Biotechnological Applications of Enzymes

Cleavage site

211

5'--G--A--A--T--T--C -- 3' Eco Rl 3'--C--T--T--A--A--G-- 5'

Symmetry axis Cleavage site

5'--G 3'--C--T--T--A--A

+

A--A--T--T--C -- 3' G-- 5'

The sequences which are recognized by these enzymes contain a two fold axis of symmetry the two strands in these regions are related by C2 axis-a 180° rotation around the axis (shown by a dot). GAATTC sequence is recognized by the enzyme and both strands of foreign DNA are cleaved to give staggered ends. SCHEME 5.25

A piece of DNA formed by the action of one restriction enzyme can be further specifically cleaved into smaller fragments by another restriction enzyme. The pattern of these fragments serves as a fingerprint of a DNA molecule. (E) Gaps in DNA are sealed by DNA ligases Certain nicks in duplex DNA can be sealed by an enzyme-DNA ligase which generates a phosphodiester bond between a 5-phosphoryl group and a directly adjacent 3-hydroxyl, using either ATP or NAD+ as an external energy source. The overall process is represented (eq I, Scheme 5.26)

DNA(nicked) + NAD

5'

+

DNA ligase 3'

DNA(sealed) + NMN + AMP (I) O O--P--O O

sealed DNA

-

+

3'

O O OH P O O

+ NAD

5'

+

DNA ligase

+ AMP + NMN

+

nick to be sealed in DNA

SCHEME 5.26

The mechanism of action of DNA ligase to seal the nick is represented (Scheme 5.27) which shows the formation of a phosphodiester linkage at the nick in DNA. In the first step the lysine side chain on the enzyme attacks (nucleophilic) NAD+ to form an AMP-DNA ligase intermediate with a phosphoamide bond generating NMN+ (nicotinamide mononucleotide). In the second step an oxygen atom of the free 5-phosphate group of the DNA attacks the phosphate group of the AMP-enzyme complex to give ADP-DNA intermediate. In the last step 3 the nucleophilic 3-hydroxyl group on the terminal residue of the adjacent DNA strand attacks the activated 5 phosphate group of ADP-DNA complex to release AMP and to form a phosphodiester

212

Bioorganic, Bioinorganic and Supramolecular Chemistry

5' DNA H 2C H (CH2)4--NH2 O

Step 1

+

O H

3'

B H H

Lys

(CH2)4--NH2 -O O OO N

+

NMN

+

Lys

H

O

P--O O

OH OO

P--O--P O O

NH2 O

Adenine

O H

CH2 H

-O --P O

Nick to be sealed

Adenine

O H

CH2 H2C H H H OH NAD

+

H H H

H OH

H 2C H H O

O H H

B

H

H OH

OH

H

OH

OH

enzyme DNA ligase

O

O

+

AMP-DNA-ligase intermediate (a) Step 2

3' DNA

Ad--O--P--O--P--O--R--N OO-

5' DNA 5' DNA H 2C

Lys

H 2C O B H H H O B H H

step 3 Adenine Lys

+

O H

3'

B H H

H

(CH2)4--NH2

H

H O

H

(CH2)4--NH2 OO O-

O H O

-O --P

Nick is sealed

P--O --P O O

Nick getting sealed

O H 2C H H O 3' DNA

Sealed DNA strand

O

O H

CH2 H2C H

O H O H H

B

5'

H

H

H OH

H

H

enzyme liberated

OH

3' DNA

ADP- DNA intermediate

O +

­

O--P--O--AD O AMP

­

SCHEME 5.27

Biotechnological Applications of Enzymes

213

linkage which seals the nick in the DNA strand. This overall detailed process may be represented in a short hand way (Scheme 5.28).

O E--(Lys)--NH2

lysine residue an enzyme

O

+

H

Step 1 +

O

+

Ad--O--P--O--P--O--R--N O+

E--(Lys)--N --P--O--Ad+NMN H O(a) AMP-DNA-ligase (intermediate)

O-

NAD (coenzyme) (simplified form)

H

+

O

Step

E--(Lys)--N --P--O--Ad + H

(a)

O-

O O OH P O O

nicked DNA

2

O OH O O

-

O P O

-

+ E(Lys)--NH2 (enzyme)

O P

O--Ad

ADP-DNA (intermediate)

Step

O O--P--O O sealed DNA

-

+ H

+

O

-

O OH O O Ad

-

O P O

-

3

+

O-- P --O--Ad O

-

O P O

AMP

SCHEME 5.28

In summary role of restriction enzymes and DNA ligase to form recombinant DNA molecules is central. The DNA molecule is split at a unique site by using a restriction enzyme (e.g., Eco RI). The DNA fragment thus produced can be annealed/joined using DNA ligase. The DNA ligase requires a free 3 OH group and a 5-phosphate group. This leads to new combinations of unrelated genes, which are introduced into suitable cells. Thus a recombinant DNA molecule contains unrelated genes. (F) The Recombinant DNA Technique In an example of recombinant DNA technology, one begins with certains circular DNA molecules found in the cells of the bacteria Escherichia coli. These molecules, called plasmids (I, Scheme 5.29), consist of double-stranded DNA arranged in a ring. Restriction enzymes cleave DNA molecules at specifric locations (a different location for each enzyme). For example, one of these enzymes may split a double-stranded DNA as shown (Scheme 5.29).

214

H--GAATTC--H enzyme H--CTTAAG--H

Bioorganic, Bioinorganic and Supramolecular Chemistry

H--G H--CTTAA

+

AATTC--H G--H

H is human gene which makes e.g., insulin

B--GAATTC--B B--CTTAAG--B

enzyme

B--G B--CTTAA

+

AATTC--B G--B

B is a plasmid from bacteria (shown in linear form for clarity)

(I)

H--G H--CTTAA

+

AATTC--B enzyme G--B

H--GAATTC--B H--CTTAAG--B

A modified plasmid (recombinant DNA molecule with a desired gene

Recombinant DNA technology.

SCHEME 5.29

A plasmid (B) is cut by the specific restriction enzyme at the restriction site. In this cut one can fit e.g., a human gene (H) which is responsible for making insulin using the enzyme DNA ligase to give a modified (recombinant) DNA molecule (Scheme 5.29). In summary the following steps are involved to produce insulin by recombinant technology : · A plasmid (a circular DNA molecule) from a bacterium e.g., E coli is cut using a specific restriction enzyme to produce a double stranded chain (Scheme 5.29/5.30) with sticky ends since each of the strands has several free bases which are ready to pair up with a complementary strip (section) of gene (i.e., this gene must be a strip of double stranded DNA that has necessary base sequence. This e.g., can be a human gene which makes insulin. · The gene strip which is to be inserted into the cut DNA (Scheme 5.30) can be made in two ways : 1. In a laboratory by chemical synthesis ; that is, chamists can combine the nucleotides in the proper sequence to make the gene. 2. One may cut a human chromosome with the same restriction enzyme. As it is the same enzyme, it cuts the human gene so as to leave the same sticky ends : · The gene strip (cut gene for insulin) and the cut DNA are mixed in the presence of a DNA ligase which joins the sticky ends and the cut DNA gets converted into a circle (plasmid) once again which now contains the desired gene. The modified plasmid (which does all things that DNA does) is then introduced into a bacterial cell, where it replicates. All these cells now generate human insulin thus one can use bacteria as a factory to manufacture specific proteins. This new technology has tremendous potential for lowering the price of drugs that are now manufactured by isolation from human or animal tissues. Not only bacteria but also plant cells can be used. Provided recombinant DNA techniques can be applied to humans and not just to bacteria, genetic diseases can be cured by this powerful technology. For instance, an infant or fetus who is missing a gene might be given that gene. Once in the cells, the gene would reproduce itself and perform for the individual's lifetime.

Biotechnological Applications of Enzymes

215

Bacterium

GA A CTT

T

TC G AA

Plasmid Foreign DNA to be cloned [restriction enzyme (EcoRI)]

restriction enzyme

G CT TAA

Sticky ends

G CTTA

Cut DNA

Cut gene for insulin

TC AAT

G

G CTT A

G

A A T T C CT TA A

G

C GA A T T

C T T A

A

Recombinant DNA

G

PROBLEMS AND EXERCISES

5.1. Write a short note on the role of enzymes in food processing taking an example from cheese making. 5.2. How enzymes are immobilized? Give their role in the production of syrups from corn starch? 5.3. How L-amino acids are prepared from their racemic mixture using immobilized enzymes? 5.4. How the level of cholesterol is controlled in humans? Explain briefly the pathway for biosynthesis of cholesterol and one typical enzyme involved. 5.5. How enzymes are used to detect heart problem? 5.6. What are genetic diseases? Give one example with the enzyme deficiency to cause it.

A

A

DNA ligase modified DNA with insulin gene inserted

plasmid enters new bacterium

Synthesis of desired protein

SCHEME 5.30

216

Bioorganic, Bioinorganic and Supramolecular Chemistry

5.7. How a sulfa drug is incorporated into an enzyme to produce nonfunctional folic acid? Explain. 5.8. What bases are required to repair the demaged portion of the DNA molecule.

--A --G --T --T --A --A --G A-- A-- C-- T-- T-- C-- T--

5.9. Compare hydrogen bonding in the -helix of proteins to that in double helix of DNA. 5.10. Write a short note on the role of restriction enzymes (endonucleases). What are stickly ends? 5.11. EcoRI restriction endonuclease recognizes the sequence GAATTC and cuts it between G and A. What will be the stickly ends of the following double-helical sequence when EcoRI acts on it?

CAAAGAATTCG GTTTCTTAAGC

5.12. Recombinant DNA technology is based on isolation of DNA, its cleavage at particular sequences, ligation of DNA fragments and their incorporation into host cells. Give the mechanism of any of these techniques. 5.13. Two different restriction endonucleases act on the following sequence of a double-stranded DNA :

The endonuclease EcoRI is specific for the sequenc GAATTC and cuts the sequence between G and A. The other endonuclease, TaqI, recognizes the sequence TCGA and cuts the sequence between T and C. What stickly ends will be created by these endonucleases? 5.14. In forming recombinant DNA molecules it may be necessary to control the enzymatic replication of DNA. How it can be achieved?

SELECTED ANSWERS TO PROBLEMS AND EXERCISES

5.8. C pairing with G and G pairing with C. 5.9. In the -helix the hydrogen bonds form between carbonyl oxygen of one residue and amide hydrogen four residues away. These hydrogen bonds are roughly parallel to the axis of the helix and involve the atoms in the backbone. The amino acid side chains which point away from the backbone are not involved in intrahelical hydrogen bonding.

Biotechnological Applications of Enzymes

217

In the case of double stranded DNA. The sugar-phosphate backbone is not involved in hydrogen bonding. However, two or three hydrogen bonds which are roughly perpendicular to the axis of the helix involve complimentary bases in opposite strands. In the -helix the cumulative effect of all the hydrogen bonds tends to stabilize the helical structure particularly within the hydrophobic interior of the protein where water does not compete for hydrogen bonding. On the other hand in DNA the major role of hydrogen bonding is to allow one strand to be complementary to the other. The helix in DNA no doubt is stabilized by hydrogen bonds between complimentary bases, however, the helix gets, major setability from stacking interactions between base pairs in the hydrophobic interior. 5.13.

EcoRI AATG TTACTTAA AATGAATT TTACTTAAGC AATTCGAGGC GCTCCG CGAGGC TCCG

Taql

5.14. The synthesis of a new strand of DNA is achieved by the successive addition of nucleotides to the end of the growing chain. DNA polymerase synthesizes DNA by the addition of one nucleotide at a time to the 3-end of the newly synthesized DNA. The nucleotide substrate is a deoxyribonucleoside 5-triphosphate (dNTP). The specific nucleotide is determined by Watson-Crick base pairing to the template strand i.e. adenine (A) pairs with thymine (T) and guanine (G) pairs with cytosine (C). The DNA synthesis can be terminated by using Sanger's method by employing 2, 3-dideoxynucleoside triphosphates (dd NTP) which differ from dNTP by lacking a 3-OH group. The ddNTP serves as a substrate for DNA polymerase. The addition of this analog blocks further growth of the new chain because it lacks the 3-hydroxyl terminus needed to form the next phosphodiester bond. Hence, fragments of various lengths are formed in which the dideoxy analog is at the 3 end (In addition to the four dNTP's the incubation mixture contains 2, 3-dideoxy analog of one of them

DNA to be sequenced

3¢----GAATTCGCTAATGC-------- 5¢----CTTAA Base P -- P -- P OCH2 H H

Primer

O H

DNA polymerase I Labeled dATP, dTTP, dCTP, dGTP Dideoxy analog of dATP A

3¢----GAATTCGCTAATGC-------- H 5¢----CTTAAGCGATT A + 3¢----GAATTCGCTAATGC-------- 5¢----CTTAAGCG A New DNA strands are separated and electrophoresed

H

ddNTP

H

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