ENZYME ENGINEERING

Immobilization of Enzymes

Many enzymes secreted by microorganisms are available on a large scale and there is no effect on their cost if they are used only once in a process. In addition, many more enzymes are such that they affect the cost and could not be economical if not reused. Therefore, reuse of enzymes led to the development of immobilization techniques. It involves the conversion of water soluble enzyme protein into a solid form of catalyst by several methods (see 17.4.2). It is only possible to immobilize microbial cells by similar techniques (Bull et al, 1983).

 

Thus, immobilization is "the imprisonment of an enzyme in a distinct phase that allows exchange with, but is separated from the bulk phase in which the substrate, effector or inhibitor molecules are dispersed and monitored" (Trevan, 1980). Imprisonment refers to arresting the enzyme by certain means where polymer matrix is formed. The first commercial application of immobilized enzyme technology was realized in 1969 in Japan with the use of Aspergillus oryzae amino acylase for the industrial production of L-amino acids. Consequently, pilot plant processes were introduced for 6-amino penicillanic acid (6 APA) production from penicillin G and for glucose to fructose conversion by immobilized glucose isomerase.

 

Advantages of Using Immobilized Enzymes

The advantages of using immobilized enzymes are : (i) reuse (ii) continuous use (iii) less labor intensive (iv) saving in capital cost (v) minimum reaction time (vi) less chance of contamination in products, (vii) more stability (viii) improved process control and (ix) high enzyme : substrate ratio.

 

The first immobilized enzymes to be scaled up to pilot plant level and industrial manufacture were immobilized amino acid acylase, panicillin G-acylase and glucose isomerase. Some other industrially important enzymes are aspartase, esterase and nitrilase.

 

Methods of Enzyme Immobilization

There are five different techniques of immobilizing enzymes : (i) adsorption, (ii) covalent bonding, (iii) entrapment, (iv) copolymerization or cross-linking, and (v) encapsulation (Fig. 17.1). For the purpose of immobilization of enzymes carriers i.e. the support materials such as matrix system, a membrane or a solid surface are used.

Adsorption

An enzyme may be immobilized by bonding to either external or internal surface of a carrier or support such as mineral support (aluminium oxide, clay), organic support (starch), modified sapharose and ion exchange resins. Bonds of low energy are involved e.g. ionic interactions, hydrogen bonds, van der Waals forces, etc. If the enzyme is immobilized externally, the carrier particle size must be very small in order to achieve a appreciable surface of bonding. These particles may have diameter ranging from 500 A to about 1 mm. Due to immobilization of enzymes on external surface, no pore diffusion limitations are encountered.

In addition, the enzyme immobilized on an internal surface is protected from abrasion, inhibitory bulk solutions and microbial attack, and a more stable and active enzyme system may be achieved. Moreover, in internal pore immobilization the pore diameters of carriers may be optimized for internal surface immobilization.

Covalent bonding

Covalent bond is formed between the chemical groups of enzyme and chemical groups on surface of carrier. Covalent bonding is thus utilized under a broad range of pH, ionic strength and other variable conditions. Immobilization steps are attachment of coupling agent followed by an activation process, or attachment of a functional group and finally attachment of the enzyme.

Different types of carriers are used in immobilization such as carbohydrates proteins and amine-bearing carriers, inorganic carriers, etc. Covalent attachment may be directed to a specific group (e.g. amine, hydroxyl, tyrosyl, etc.) on the surface of the enzyme. Hydroxyl and amino groups are the main groups of the enzymes with which it forms bonds, whereas sulphydryl group least involved.

Entrapment

Enzymes can be physically entrapped inside a matrix (support) of a water soluble polymer such as polyacrylamide type gels and naturally derived gels e.g. cellulose triacetate, agar, gelatin, carrageenan, alginate, etc. (Fig. 17.1C). The form and nature of matrix vary. Pore size of matrix should be adjusted to prevent the loss of enzyme from the matrix due to excessive diffusion. There is possibility of leakage of low molecular weight enzymes from the gel. Agar and carrageenan have large pore sizes (< 10).

 

There are several methods for enzyme entrapment: (i) inclusion in gels (enzyme entrapped in gels), (ii) inclusion in fibers (enzyme entrapped in fiber format), and (iii) inclusion in microcapsules (enzymes entrapped in microcapsules formed monomer mixtures such as polyamine and polybasic chloride, polyphenol and polyisocyanate). The entrapment of enzymes has been widely used for sensing application, but not much success has been achieved with industrial process.

Cross-linking or Co-polymerization

Cross-linking is characterized by covalent bonding between the various molecules of an enzyme via a polyfunctional reagent such as glutaraldehyde, diazonium salt, hexamethylene disocyanate, and N-N' ethylene bismaleimide. The demerit of using polyfunctional reagents is that they can denature the enzyme. This technique is cheap and simple but not often used with pure proteins because it produces very little of immobilized enzyme that has very high intrinsic activity. It is widely used in commercial preparation.

Encapsulation

Encapsulation is the enclosing of a droplet of solution-of enzyme in a semipermeable membrane capsule. The capsule is made up of cellulose nitrate and nylon. The method of encapsulation is cheap and simple but its effectiveness largely depends on the stability of enzyme although the catalyst is very effectively retained within the capsule. This technique is restricted to medical sciences only (Fig. 17.1D).

In this method a large quantity of enzyme is immobilized but the biggest disadvantage is that only small substrate molecule is utilized with the intact membrane. Chent (1977) has given the method of enzyme encapsulation.

APPLICATIONS OF ENZYMES

This list contains some of products of enzyme biotechnology you might use everyday in your own home. In many cases, the commercial processes first exploited naturally occurring enzymes. However, this does not mean the enzyme(s) being used were as efficient as they could be. With time, research, and improved protein engineering methods, many enzymes have been genetically modified to be more effective at the desired temperatures, pH, or under other manufacturing conditions typically inhibitory to enzyme activity (eg. harsh chemicals), making them more suitable and efficient for industrial or home applications.

• Stickies Removal
  Enzymes are used by the pulp and paper industry for the removal of “stickies”, the glues, adhesives and coatings that are introduced to pulp during recycling of paper. Stickies are tacky, hydrophobic, pliable organic materials that not only reduce the quality of the final paper product, but can clog the paper mill machinery and cost hours of downtime. Chemcial methods for removal of stickies have, historically, not been 100% satisfactory.
  Stickies are held together by ester bonds, and the use of esterase enzymes in pulp has vastly improved their removal. Esterases cut the stickies into smaller, more water soluble compounds, facilitating their removal from the pulp. Since the early half of this decade, esterases have become a common approach to stickies control. Their limitations are, being enzymes, they are typically only effective at moderate temperature and pH. Also, certain esterases might only be effective against certain types of esters and the presence of other chemicals in the pulp can inhibit their activity. The search is on for new enzymes, and genetic modifications of existing enzymes, to broaden their effective temperature and pH ranges, and substrate capabilities.
• Detergents
  Enzymes have been used in many kinds of detergents for over 30 years, since they were first introduced by Novozymes. Traditional use of enzymes in laundry detergents involved those that degrade proteins causing stains, such as those found in grass stains, red wine and soil. Lipases are another useful class of enzymes that can be used to dissolve fat stains and clean grease traps or other fat-based cleaning applications.
  Currently, a popular area of research is the investigation of enzymes that can tolerate, or even have higher activities, in hot and cold temperatures. The search for thermotolerant and cryotolerant enzymes has spanned the globe. These enzymes are especially desirable for improving laundry processes in hot water cycles and/or at low temperatures for washing colors and darks. They are also useful for industrial processes where high temperatures are required, or for bioremediation under harsh conditions (eg. in the arctic). Recombinant enzymes (engineered proteins) are being sought using different DNA technologies such as site-directed mutagenesis and DNA shuffling.
• Textiles
  Enzymes are now widely used to prepare the fabrics that your clothing, furniture and other household items are made of. Increasing demands to reduce pollution caused by the textile industry has fueled biotechnological advances that have replaced harsh chemicals with enzymes in nearly all textile manufacturing processes. Enzymes are used to enhance the preparation of cotton for weaving, reduce impurities, minimize “pulls” in fabric, or as pre-treatment before dying to reduce rinsing time and improve colour quality. All of these steps not only make the process less toxic and eco-friendly, they reduce costs associated with the production process, and consumption of natural resources (water, electricity, fuels), while also improving the quality of the final textile product.
• Foods and Beverages
  This is the domestic application for enzyme technology that most people are already familiar with. Historically, humans have been using enzymes for centuries, in early biotechnological practices, to produce foods, without really knowing it. It was possible to make wine, beer, vinegar and cheeses, for example, because of the enzymes in the yeasts and bacteria that were utilized.
  Biotechnology has made it possible to isolate and characterize the specific enzymes responsible for these processes. It has allowed the development of specialized strains for specific uses that improve the flavour and quality of each product. Enzymes can also be used to make the process cheaper and more predictable, so a quality product is ensured with every batch brewed. Other enzymes reduce the length of time required for aging, help clarify or stabilize the product, or help control alcohol and sugar contents.
  For years, enzymes have also been used to turn starch into sugar. Corn and wheat syrups are used throughout the food industry as sweeteners. Using enzyme technology, the production of these sweeteners can be less expensive than using sugarcane sugar. Enzymes have been developed and enhanced using biotechnological methods, for every step of the process.

 Leather
In the past, the process of tanning hides into useable leather involved the use of many harmful chemicals. Enzyme technology has advanced such that some of these chemicals can be replaced and the process is actually faster and more efficient. There are enzymes that can be applied to the first steps of the process where fat and hair are removed from the hides. Enzymes are also used during cleaning, and keratin and pigment removal, and to enhance the softness of the hide. They also help stabilize the leather during the tanning process to prevent it from rotting.

 Biodegradable plastic
Plastics manufactured by traditional methods come from non-renewable hydrocarbon resources. They consist of long polymer molecules that are tightly bound to one another and cannot be broken down easily by decomposing microorganisms. Biodegradable plastics can be made using plant polymers from wheat, corn or potatoes, and consist of shorter, more easily degraded polymers.
Since biodegradable plastics are more water soluble, many current products that contain them are a mixture of biodegradable and non-degradable polymers. Certain bacteria can produce granules of plastic within their cells. The genes for enzymes involved in this process have been cloned into plants which can produce the granules in their leaves. The cost of plant-based plastics limits their use, and they have not met with widespread consumer acceptance.

 Bioethanol
Bioethanol is a biofuel that has already met with widespread public acceptance. You might already be using bioethanol when you add fuel to your vehicle. Bioethanol can be produced from starchy plant materials using enzymes capable of efficiently making the conversion. At present, corn is a widely used source of starch, however increasing interest in bioethanol is raising concerns as corn prices rise and corn as a food supply is being threatened. Other plants including wheat, bamboo, or other grasses are possible candidate sources of starch for bioethanol production.
It is debatable whether the cost of making bioethanol is less than for the consumption of fossil fuels, in terms of greenhouse emissions. Bioethanol production (growing crops, shipping, manufacturing) still requires a large input of non-renewable resources. Technological research and manipulation of enzymes to make the process more efficient, thus requiring less plant material or consuming less fossil fuels, are in the works, to improve on this area of biotechnology.

Therapeutic Uses

Enzymes are used for this purpose where some inborn errors of metabolism occur due to missing of enzyme (see Monoclonal antibodies) where specific genes are introduced to encode specific missing enzymes. However, in most of cases certain diseases are treated by administering the appropriate enzyme. For example, virilization of a disease developed due to loss of an hydroxylase enzyme from adrenal cortex and introduction of hydroxyl group (-OH) on 21-carbon of ring structure of steroid hormone. Steroids are compounds having a common skeleton in the form of perhydro-1, 2-cyclo-pentano-phenanthrene (Fig. 17.4). The missing enzyme synthesizes aldosterone (male hormone) in excess leading to masculinization of female baby and precocious sexual activity in males in about 5-7 years.

 

Similarly, treatment of leukaemia (a disease in which leukaemic cells require exogenous asparagine for their growth) could be done by administering asparaginase of bacterial origin.

In dairy industry

For a long time calf rennet has been used in dairy industry. In recent years, calf rennets are replaced by microbial rennets (e.g. Mucor michei). They are acid aspartate proteases. They slightly differ from calf rennets as they depend for reaction with casein on Ca++, temperature, pH, etc.

 

Lactase (produced by Bacillus stearothermophilus) is used for hydrolysis of lactose in whey or milk, and lipase for flavor development in special cheeses.

In brewing industry

Enzymes used in brewing industry are -amylase, -glucanase and protease which are required for malt in substitution of barley. Source of these enzymes is B. amyloliquefaciens. -amylase is not required for liquefaction or brewing adjuncts and -glucanase alleviates filtration problems due to poor malt quality and neutral protease helps in the inhibition of alkaline protease by an inhibitor.

 

In wine industry

Pectic enzymes are used in wine industry for high yield of products of improved quality. The pectic enzymes are pectin transeliminase (PTE), polymethyl galacturonase (PMG), polygalacturonase (PG),pectine esterase (PE), etc. However, pectic enzymes give a good result when combined with other enzymes e.g. protease glucoamylase, etc.

In pharmaceutical industry

Penicillin G/V acylase, glucose isomerase, etc. are widely used in Pharmaceuticals for the production of semisynthetic penicillins and fructose syrup, respectively. All penicillins consist of an active beta lactam ring i.e. 6-amine penillanic acid (6 APA) group combined with different side chains (R group) (Fig. 16.2) Penicillin G/V acylase removes G/V group from penicillin G/V resulting in separation of 6 APA and R groups

Industrial applications

Enzymes are used in the chemical industry and other industrial applications when extremely specific catalysts are required. However, enzymes in general are limited in the number of reactions they have evolved to catalyze and also by their lack of stability in organic solvents and at high temperatures. As a consequence, protein engineering is an active area of research and involves attempts to create new enzymes with novel properties, either through rational design or in vitro evolution.[91][92] These efforts have begun to be successful, and a few enzymes have now been designed "from scratch" to catalyze reactions that do not occur in nature.[93]

Application	Enzymes used	Uses
Food processing Amylases catalyze the release of simple sugars from starch.	Amylases from fungi and plants	Production of sugars from starch, such as in making high-fructose corn syrup.[94] In baking, catalyze breakdown of starch in the flour to sugar. Yeast fermentation of sugar produces the carbon dioxide that raises the dough.
	Proteases	Biscuit manufacturers use them to lower the protein level of flour.
Baby foods	Trypsin	To predigest baby foods
Brewing industry Germinating barley used for malt	Enzymes from barley are released during the mashing stage of beer production.	They degrade starch and proteins to produce simple sugar, amino acids and peptides that are used by yeast for fermentation.
	Industrially produced barley enzymes	Widely used in the brewing process to substitute for the natural enzymes found in barley.
	Amylase, glucanases, proteases	Split polysaccharides and proteins in the malt.
	Betaglucanases and arabinoxylanases	Improve the wort and beer filtration characteristics.
	Amyloglucosidase and pullulanases	Low-calorie beer and adjustment of fermentability.
	Proteases	Remove cloudiness produced during storage of beers.
	Acetolactatedecarboxylase (ALDC)	Increases fermentation efficiency by reducing diacetyl formation.[95]
Fruit juices	Cellulases, pectinases	Clarify fruit juices.
Dairy industry Roquefort cheese	Rennin, derived from the stomachs of young ruminant animals (like calves and lambs)	Manufacture of cheese, used to hydrolyze protein
	Microbially produced enzyme	Now finding increasing use in the dairy industry
	Lipases	Is implemented during the production of Roquefort cheese to enhance the ripening of the blue-mold cheese.
	Lactases	Break down lactose to glucose and galactose.
Meat tenderizers	Papain	To soften meat for cooking
Starch industry Glucose Fructose	Amylases, amyloglucosideases and glucoamylases	Converts starch into glucose and various syrups.
	Glucose isomerase	Converts glucose into fructose in production of high-fructose syrups from starchy materials. These syrups have enhanced sweetening properties and lower calorific values than sucrose for the same level of sweetness.
Paper industry A paper mill in South Carolina	Amylases, Xylanases, Cellulases and ligninases	Degrade starch to lower viscosity, aiding sizing and coating paper. Xylanases reduce bleach required for decolorizing; cellulases smooth fibers, enhance water drainage, and promote ink removal; lipases reduce pitch and lignin-degrading enzymes remove lignin to soften paper.
Biofuel industry Cellulose in 3D	Cellulases	Used to break down cellulose into sugars that can be fermented (see cellulosic ethanol)
	Ligninases	Use of lignin waste
Biological detergent	Primarily proteases, produced in an extracellular form from bacteria	Used for presoak conditions and direct liquid applications helping with removal of protein stains from clothes
	Amylases	Detergents for machine dish washing to remove resistant starch residues
	Lipases	Used to assist in the removal of fatty and oily stains
	Cellulases	Used in biological fabric conditioners
Contact lens cleaners	Proteases	To remove proteins on contact lens to prevent infections
Rubber industry	Catalase	To generate oxygen from peroxide to convert latex into foam rubber
Photographic industry	Protease (ficin)	Dissolve gelatin off scrap film, allowing recovery of its silver content.
Molecular biology Part of the DNA double helix	Restriction enzymes, DNA ligase and polymerases	Used to manipulate DNA in genetic engineering, important in pharmacology, agriculture and medicine. Essential for restriction digestion and the polymerase chain reaction. Molecular biology is also important in forensic science.

Methods of Purification of Enzymes

Enzymes are manufactured in bioreactors for commercial use. These enzymes are in the crude form and have to be purified for further use. The extraction methods are followed by the purification processes. There are mainly three major purification methods depending on the technique or property of enzyme.

1. Based on ionic properties of enzymes
2. Based on the ability to get adsorbed
3. Based on difference in size of molecules

1. Techniques depending on the ionic properties of enzymes

a. Salting out
It is done by varying the pH of the solution or by addition of chemical agents which carry out precipitation.
At the isoelectric pH (pI), proteins have minimum solubility and hence get precipitated out as pure crystals. For example the digestive enzyme pepsin is purified by this process.
Salting out is also possible by addition of chemicals such as Ammonium sulphate, Acetone etc.

b. Electrophoresis

It is the movement of charged particles under the influence of an electric field. The ions migrate based on the electric charge and strength of the field. The velocity of migration of ions in the electric field is given by

Ve = qE / 6π μcrp
q- Charge on the particle
E- Electrical potential
μc - viscosity of the liquid
rp - radius of the particle

Hence the velocity of migration of charged particle is proportional to the charge since all other values of the equation are constants.

c. Ion exchange chromatography

Selective adsorption and exchange of ions take place in the adsorption sites of ion exchange columns filled with ion exchange resins.

Eg: DEAE (Diethyl Amino Ethyl) Cellulose is an Anion exchanger and DM (Dimethyl) Cellulose is a Cation exchanger.

2. Techniques depending on the adsorbing properties of enzymes

a. Adsorption chromatography

It is based on the principle of adsorption. Solute/ enzyme get adsorbed onto the particular sites of the adsorption chromatographic column depending on the effective distribution coefficient. These are then eluted using various solvents. The packing materials used include starch, diatomaceous earth etc.
Effective distribution coefficient is the ratio of distribution of solute across the different phases of chromatography. The process is widely used for initial recovery of extra cellular enzymes manufactured on a large scale.

b. Affinity chromatography

This technique makes use of enzyme substrate interactions. A matrix with a ligand is packed in the column. As the enzyme solution pass through the column, the solute/enzyme molecules get attached to the ligand. These are then eluted by suitable eluants/solvents. In effect the Matrix ligand enzyme complexes remain adsorbed to the column till it gets eluted.

3. Techniques depending on the size of enzymes

a. Molecular sieve/ Gel filtration/ Gel permeation Chromatography

This makes use of gel material as the supporting matrix. Smaller particles get entrapped in the pores whereas larger particles get through the interfacial space. Hence the larger particles get separated first followed by smaller particles

b. Ultra filtration (Dialysis)

It is the process of passage of solvent molecules from a region of higher concentration to a region of lower concentration through a semi permeable membrane. The technique is used for purification of crude enzymes. It is used for enzyme concentration as well as enzyme purification.

Enzymes when present in aqueous solutions cannot be retained in the bioreactor. They can be retained in the system by the process of immobilization. It is the process of attaching a cell/ enzyme to an insoluble inert support. The purity of such immobilized enzymes is higher than free enzymes. The turn over number is high and enzyme activity can be retained for a longer duration.

Other sophisticated methods such as electro dialysis, electro filtration, isoelectric focusing, forced flow electrophoresis, electro decantation and isotachophoresis are also developed and used. Membrane based processes such as reverse osmosis and pervaporation are also gaining popularity. For membrane processes, retained species tend to concentrate on the upstream of the membrane and reduce the permeation flux in a process called concentration polarization.

Enzyme assay

Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.

Types of assay

All enzyme assays measure either the consumption of substrate or production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments:[3]

• Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess of substrate, the approximation that the amount of free substrate is approximately equal to the amount of the initial substrate can be made. The initial rate experiment is the simplest to perform and analyze, being relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most commonly used type of experiment in enzyme kinetics.

• Progress curve experiments. In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. We note in passing that, while they are less common now, progress curve experiments were widely used in the early period of enzyme kinetics.

• Transient kinetics experiments. In these experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require specialist techniques (such as flash photolysis of caged compounds) or rapid mixing (such as stopped-flow, quenched flow or continuous flow).

• Relaxation experiments. In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, although they can be under appropriate conditions.

Enzyme catalysis

Enzyme catalysis is the catalysis of

chemical reactions by specialized proteins known as enzymes. Catalysis of biochemical reactions in the

cell is vital due to the very low reaction rates of the uncatalysed reactions.[citation needed]

The mechanism of enzyme catalysis is similar in principle to other types of chemical catalysis. By providing an alternative reaction route the enzyme reduces the energy required to reach the highest energy transition state of the reaction. The reduction of activation energy (Ea) increases the number of reactant molecules with enough energy to reach the activation energy and form the product

Induced fit

The favored model for the enzyme-substrate interaction is the induced fit model.[1] This model proposes that the initial interaction between enzyme and substrate is relatively weak, but that these weak interactions rapidly induce conformational changes in the enzyme that strengthen binding.

[edit] Mechanisms of an alternative reaction route

These conformational changes also bring catalytic residues in the active site close to the chemical bonds in the substrate that will be altered in the reaction. After binding takes place, one or more mechanisms of catalysis lowers the energy of the reaction's

transition state, by providing an alternative chemical pathway for the reaction. There are six possible mechanisms of "over the barrier" catalysis as well as a "through the barrier" mechanism:

[edit] Bond strain

This is the principal effect of induced fit binding, where the affinity of the enzyme to the transition state is greater than to the substrate itself. This induces structural rearrangements which strain substrate bonds into a position closer to the conformation of the transition state, so lowering the energy difference between the substrate and transition state and helping catalyze the reaction.

However, the strain effect is, in fact, a ground state destabilization effect, rather than transition state stabilization effect.[3][4] Furthermore, enzymes are very flexible and they cannot apply large strain effect.[5]

[edit] Proximity and orientation

This increases the rate of the reaction as enzyme-substrate interactions align reactive chemical groups and hold them close together. This reduces the entropy of the reactants and thus makes reactions such as ligations or addition reactions more favorable, there is a reduction in the overall loss of entropy when two reactants become a single product.

This effect is analogous to an effective increase in concentration of the reagents. The binding of the reagents to the enzyme gives the reaction intramolecular character, which gives a massive rate increase.

[edit] Proton donors or acceptors

Proton donors and acceptors, i.e. acids and

base may donate and accept protons in order to stabilize developing charges in the transition state.This typically has the effect of activating nucleophile and electrophile groups, or stabilizing leaving groups. Histidine is often the residue involved in these acid/base reactions, since it has a pKa close to neutral pH and can therefore both accept and donate protons.

It is important to clarify that the modification of the pKa’s is a pure part of the electrostatic mechanism.[4] Furthermore, the catalytic effect of the above example is mainly associated with the reduction of the pKa of the oxyanion and the increase in the pKa of the histidine, while the proton transfer from the serine to the histidine is not catalyzed significantly, since it is not the rate determining barrier.[12]

[edit] Electrostatic catalysis

Stabilization of charged transition states can also be by residues in the active site forming ionic bonds (or partial ionic charge interactions) with the intermediate. These bonds can either come from acidic or

basic side chains found on amino acids such as lysine, arginine,

aspartic acid or glutamic acid or come from metal cofactors such as zinc. Metal ions are particularly effective and can reduce the pKa of water enough to make it an effective nucleophile.

[edit] Covalent catalysis

Covalent catalysis involves the substrate forming a transient covalent bond with residues in the active site or with a cofactor. This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction. The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme. This mechanism is found in enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is formed. Schiff base formation using the free amine from a lysine residue is another mechanism, as seen in the enzyme aldolase during glycolysis.

[edit] Quantum tunneling

These traditional "over the barrier" mechanisms have been challenged in some cases by models and observations of "through the barrier" mechanisms (quantum tunneling). Some enzymes operate with kinetics which are faster than what would be predicted by the classical ΔG‡. In "through the barrier" models, a proton or an electron can tunnel through activation barriers.[19][20] Quantum tunneling for protons has been observed in tryptamine oxidation by aromatic amine dehydrogenase.[21]

[edit] Active enzyme

The binding energy of the enzyme-substrate complex cannot be considered as an external energy which is necessary for the substrate activation. The enzyme of high energy content may firstly transfer some specific energetic group X1 from catalytic site of the enzyme to the final place of the first bound reactant, then another group X2 from the second bound reactant (or from the second group of the single reactant) must be transferred to active site to finish substrate conversion to product and enzyme regeneration.[

Classification and Nomenclature of Enzymes

The main divisions and subclasses are:

Class 1. Oxidoreductases.

To this class belong all enzymes catalysing oxidoreduction reactions. The substrate that is oxidized is regarded as hydrogen donor. The systematic name is based on donor:acceptor oxidoreductase. The common name will be dehydrogenase, wherever this is possible; as an alternative, reductase can be used. Oxidase is only used in cases where O2 is the acceptor.

The second figure in the code number of the oxidoreductases, unless it is 11, 13, 14 or 15, indicates the group in the hydrogen (or electron) donor that undergoes oxidation: 1 denotes a -CHOH- group, 2 a -CHO or -CO-COOH group or carbon monoxide, and so on, as listed in the key.

Class 2. Transferases.

Transferases are enzymes transferring a group, e.g. a methyl group or a glycosyl group, from one compound (generally regarded as donor) to another compound (generally regarded as acceptor). The systematic names are formed according to the scheme donor:acceptor grouptransferase. 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.

Class 3. Hydrolases.

These enzymes catalyse the hydrolytic cleavage of C-O, C-N, C-C and some other bonds, including phosphoric anhydride bonds. Although the systematic name always includes hydrolase, 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.

A number of hydrolases acting on ester, glycosyl, peptide, amide or other bonds are known to catalyse not only hydrolytic removal of a particular group from their substrates, but likewise the transfer of this group to suitable acceptor molecules.

Class 4. Lyases.

Lyases 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. Various subclasses of the lyases include pyridoxal-phosphate enzymes that catalyse the elimination of a β- or γ-substituent from an α-amino acid followed by a replacement of this substituent by some other group.

Class 5. Isomerases.

These enzymes catalyse geometric or structural changes within one molecule. According to the type of isomerism, they may be called racemases, epimerases, cis-trans-isomerases, isomerases, tautomerases, mutases or cycloisomerases.

In some cases, 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)+.

The subclasses are formed according to the type of isomerism, the sub-subclasses to the type of substrates.

Class 6. Ligases.

Ligases are enzymes catalysing the joining together of two molecules coupled with the hydrolysis of a diphosphate bond in ATP or a similar triphosphate. The systematic names are formed on the system X:Y ligase (ADP-forming). In earlier editions of the list the term 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 emphasis the synthetic nature of the reaction). Consequently NC-IUB decided in 1983 to abandon the use of synthetase for common names, and to replace them with names of the type X-Y ligase. In a few cases in Group 6, where the reaction is more complex or there is a common name for the product, a synthase name is used.

Notes on the Factors Affecting Enzyme Activity

At a fixed substrate concentration, an increase in enzyme concentration increases the rate of an enzyme catalyzed reaction until the substrate concentration becomes the limiting factor.

When the substrate concentration is not limiting, the rate of an enzyme catalyzed reaction is directly proportional to the enzyme concentration. The rate slows down when [s] starts becoming limiting factor. The rate remains unchanged at high [E] when [s] is the limiting factor.

Effect of Substrate Concentration

At a fixed enzyme concentration, an increase in f substrate concentration increases the rate of an enzyme § catalyzed reaction until the enzyme concentration becomes the limiting factor.

When the enzyme concentration is not limiting, the rate of an enzyme catalyzed reaction is directly proportional to the substrate concentration. on increasing [s] when [E] starts becoming limiting factor, the rate of the enzyme catalyzed reaction slows down. At high [s], the rate remains unchanged because [s] becomes limiting factor.

Effect of pH

Enzymes, being proteins, are affected by the changes in the pH of the reaction medium. Most of the enzymes are, in fact, active only within a narrow range of pH, typically pH 5 to pH 9. The most favorable pH at which an enzyme activity is the maximum is known as the optimum pi I for the enzyme.

The optimum pH value varies from enzyme to enzyme. The pH affects the£ degree of ionization of the side chains of then amino acid residues of proteins and thereby their three dimensional structure. Furthermore, ionization state of amino acid side chains present in the active site of enzymes are responsible for the catalytic activity of enzymes (see later in this chapter).

The pH also affects the ionization characteristics of the substrates and coenzymes, and thereby, their binding with the enzyme. Thus pH of the reaction medium affects the catalytic activity of the enzyme. The catalytic activity of the enzyme as a function of pH usually appears as a bell-shaped curve.

Effect of Temperature

Like most of the chemical reactions, the rate of an enzyme catalyzed reaction increases with the increase in temperature of the reaction medium.

However, enzymes-being proteins are gradually denatured and lose their activity at the temperature beyond 40 - 50°C. Some enzymes, however, remain active even at the temperature as high as 100oC. Each enzyme has an optimum temperature at which its activity is the maximum.

Effect of Inhibitors and Activators

Many molecules and ions, when present in the reaction medium; affect the rate of enzyme-catalyzed reactions. These substances bind to the enzyme or the enzyme-substrate complex, thereby, affecting the rate.

Substances which lower the reaction rate are called enzyme inhibitors and substances which increase the rate are as enzyme activators. Inhibitors and activators are very important in the cellular regulation of enzymes.

Inhibitors can be classified as reversible or irreversible. Reversible inhibitors bind with the enzyme by weak noncovalent bonds and can be removed by dialysis restore the active enzyme. On the other hand irreversible inhibitors bind with enzyme by covalent bonds and cannot be removed from the enzyme by dialysis.

Mechanism of Enzyme Action

Enzymes, as explained before accelerate the rates of biochemical reactions show great deal of substrate specificity. Therefore, any theory on the mechanic enzymatic reaction must take these two facts into account.

Activation Energy

The conversion of reactants to products in any chemical reaction is accompanied by continuous change in energy. During the reaction pathway, the reactants p through a transition state.

This transition state of the reactants has higher free energy than either the ground state reactants or products.

The difference in free energy between transition state and ground state reactants is called the activation energy. Activation energy, thus, is the energy required to activate the reactants to move them to transition state.

ENZYME INHIBITION

An enzyme inhibitor is a molecule which binds to enzymes and decreases their activity. Since blocking an enzyme's activity can kill a pathogen or correct a metabolic imbalance, many drugs are enzyme inhibitors. They are also used as herbicides and pesticides. Not all molecules that bind to enzymes are inhibitors; enzyme activators bind to enzymes and increase their enzymatic activity, while enzyme substrates bind and are converted to products in the normal catalytic cycle of the enzyme.

The binding of an inhibitor can stop a substrate from entering the enzyme's active site and/or hinder the enzyme from catalyzing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically (e.g. via covalent bond formation). These inhibitors modify key amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind non-covalently and different types of inhibition are produced depending on whether these inhibitors bind to the enzyme, the enzyme-substrate complex, or both.

Reversible inhibitors

[edit] Types of reversible inhibitors

Reversible inhibitors bind to enzymes with non-covalent interactions such as hydrogen bonds, hydrophobic interactions and ionic bonds. Multiple weak bonds between the inhibitor and the active site combine to produce strong and specific binding. In contrast to substrates and irreversible inhibitors, reversible inhibitors generally do not undergo chemical reactions when bound to the enzyme and can be easily removed by dilution or dialysis.

Competitive inhibition: substrate (S) and inhibitor (I) compete for the active site.

There are four kinds of reversible enzyme inhibitors. They are classified according to the effect of varying the concentration of the enzyme's substrate on the inhibitor.[2]

In competitive inhibition, the substrate and inhibitor cannot bind to the enzyme at the same time, as shown in the figure on the left. This usually results from the inhibitor having an affinity for the active site of an enzyme where the substrate also binds; the substrate and inhibitor compete for access to the enzyme's active site. This type of inhibition can be overcome by sufficiently high concentrations of substrate (Vmax remains constant), i.e., by out-competing the inhibitor. However, the apparent Km will increase as it takes a higher concentration of the substrate to reach the Km point, or half the Vmax. Competitive inhibitors are often similar in structure to the real substrate (see examples below). In uncompetitive inhibition, the inhibitor binds only to the substrate-enzyme complex, it should not be confused with non-competitive inhibitors. This type of inhibition causes Vmax to decrease (maximum velocity decreases as a result of removing activated complex) and Km to decrease (due to better binding efficiency as a result of Le Chatelier's principle and the effective elimination of the ES complex thus decreasing the Km which indicates a higher binding affinity). In mixed inhibition, the inhibitor can bind to the enzyme at the same time as the enzyme's substrate. However, the binding of the inhibitor affects the binding of the substrate, and vice versa. This type of inhibition can be reduced, but not overcome by increasing concentrations of substrate. Although it is possible for mixed-type inhibitors to bind in the active site, this type of inhibition generally results from an allosteric effect where the inhibitor binds to a different site on an enzyme. Inhibitor binding to this allosteric site changes the conformation (i.e., tertiary structure or three-dimensional shape) of the enzyme so that the affinity of the substrate for the active site is reduced. Non-competitive inhibition is a form of mixed inhibition where the binding of the inhibitor to the enzyme reduces its activity but does not affect the binding of substrate. As a result, the extent of inhibition depends only on the concentration of the inhibitor. Vmax will decrease due to the inability for the reaction to proceed as efficiently, but Km will remain the same as the actual binding of the substrate, by definition, will still function properly.

Irreversible inhibitors

Irreversible inhibitors are substances that bond to the enzyme covalently. They are not displaced by the substrate that usually binds to the enzyme (because the substrate binds with hydrogen bonds which are much weaker than covalent bonds). By bonding to the enzyme, either at the active site (so the substrate cannot bind there) or at any other part of the enzyme, the inhibitor may be able to change the conformation (shape) of the enzyme. It does this by either breaking hydrogen bonds or making others form in the wrong place. This changes the enzyme structure. These inhibitors are called irreversible because they do not easily leave the enzyme. The enzyme is no longer able to function.

Enzyme assay

Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of

enzyme kinetics and

enzyme inhibition.

Types of assay

Continuous assays [edit]

Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. There are many different types of continuous assays.

Spectrophotometric [edit]

In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, and these are called colorimetric assays. The MTT assay, a redox assay using a tetrazolium dye as substrate is an example of a colorimetric assay.

UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do not in their oxidized forms. An oxidoreductase using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at a wavelength of 340 nm as it consumes the coenzyme.[4]

Fluorometric [edit]

Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light.

Calorimetric [edit]

Chemiluminescence of Luminol

Calorimetry is the measurement of the heat released or absorbed by chemical reactions. These assays are very general, since many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is required. These assays can be used to measure reactions that are impossible to assay in any other way.[6]

Chemiluminescent [edit]

Chemiluminescence is the emission of light by a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation. These types of assay can be extremely sensitive, since the light produced can be captured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a reaction will be detected.

Light Scattering [edit]

Static light scattering measures the product of weight-averaged molar mass and concentration of macromolecules in solution. Given a fixed total concentration of one or more species over the measurement time, the scattering signal is a direct measure of the weight-averaged molar mass of the solution, which will vary as complexes form or dissociate. Hence the measurement quantifies the stoichiometry of the complexes as well as kinetics. Light scattering assays of protein kinetics is a very general technique that does not require an enzyme.

Microscale Thermophoresis [edit]

Microscale Thermophoresis (MST)[7] measures the size, charge and hydration entropy of molecules/substrates in real time.[8] The thermophoretic movement of a fluorescently labeled substrate changes significantly as it is modified by an enzyme. This enzymatic activity can be measured with high time resolution in real time.[9] The material consumption of the all optical MST method is very low, only 5 µl sample volume and 10nM enzyme concentration are needed to measure the enzymatic rate constants for activity and inhibition. MST allows to measure the modification of two different substrates at once (multiplexing) if both substrates are labeled with different fluorophores. Thus substrate competition experiments can be performed.

Discontinuous assays [edit]

Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption is measured in these samples.

Radiometric [edit]

Radiometric assays measure the incorporation of radioactivity into substrates or its release from substrates. The radioactive isotopes most frequently used in these assays are 14C, 32P, 35S and 125I. Since radioactive isotopes can allow the specific labelling of a single atom of a substrate, these assays are both extremely sensitive and specific. They are frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells).

Chromatographic [edit]

Chromatographic assays measure product formation by separating the reaction mixture into its components by chromatography. This is usually done by high-performance liquid chromatography (HPLC), but can also use the simpler technique of thin layer chromatography. Although this approach can need a lot of material, its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tag. Assay sensitivity has also been increased by switching protocols to improved chromatographic instruments (e.g. ultra-high pressure liquid chromatography) that operate at pump pressure a few-fold higher than HPLC instruments (see High-performance liquid chromatography#Pump_pressure).[10]

Enzyme catalysis

Enzyme catalysis is the catalysis of chemical reactions by specialized proteins known as enzymes. Catalysis of biochemical reactions in the

cell is vital due to the very low reaction rates of the uncatalysed reactions.[citation needed]

The mechanism of enzyme catalysis is similar in principle to other types of chemical catalysis. By providing an alternative reaction route the enzyme reduces the energy required to reach the highest energy transition state of the reaction. The reduction of activation energy (Ea) increases the number of reactant molecules with enough energy to reach the activation energy and form the product.

General acid/base catalysis/Proton donors or acceptors

General acid/base catalysis' rate determining step is the proton transfer step. Therefore, general acid catalysis has its reaction rate depending on all the acids present; similarly, the general base catalysis has its reaction rate depending on all the bases present. The preferred reaction environment is neutral PH for both reactions, because high concentration of H+ or OH- can damp out the catalytic contributions from other acids and bases, thus, turning the "general" acid or base reaction into "specific" acid or base catalysis.

Since the proton transfer step determines the rate of the reaction, it is important to examine the effectiveness of the general catalysis.

Covalent catalysis [edit]

Covalent catalysis involves the substrate forming a transient covalent bond with residues in the active site or with a cofactor. This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction. The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme. This mechanism is found in enzymes such as proteases like chymotrypsin and trypsin, where an acyl-enzyme intermediate is formed. Schiff base formation using the free amine from a lysine residue is another mechanism, as seen in the enzyme aldolase during glycolysis.

Electrostatic catalysis [edit]

Stabilization of charged transition states can also be by residues in the active site forming ionic bonds (or partial ionic charge interactions) with the intermediate. These bonds can either come from acidic or

basic side chains found on amino acids such as lysine, arginine,

aspartic acid or glutamic acid or come from metal cofactors such as zinc. Metal ions are particularly effective and can reduce the pKa of water enough to make it an effective nucleophile.

Metal ion catalysis

Metal ion catalysis, is a specific mechanism that utilizes metalloenzymes with tightly bound metal ions such as Fe2+, Cu2+, Zn2+, Mn2+, Co3+, Ni3+, Mo6+ (the first three being the most commonly used) to carry out a catalytic reaction. This area of catalysis also includes metal ions which are not tightly bound to a metalloenzyme, such as Na+, K+, Mg2+, Ca2+.

Enzymes can catalyze a reaction by the use of metals. Metals often facilitate the catalytic process in different ways. The metals can either assist in the catalyic reaction, activate the enzyme to begin the catalysis or they can inhibit reactions in solution. Metals activate the enzyme by changing its shape but are not actually involved in the catalytic reaction.

Nucleophilic catalysis Many enzymes binds substrates with covalent bonding. Enzymes always are nucleophilic. Substrates are electrophilic. Therefore, the enzymes attacks the electrophilic center of substrates. This reaction is very rapid.

Electrophilic catalysis The enzyme reaction can be catalyzed by removing the electron.

ALLOSTERISM

The main complication that comes from drug therapy at nicotinic receptors is the development of side effects.  This is largely due to the fact that current drugs on the market will affect many different subtypes of nicotinic receptors.  Furthermore, the main reason for this is because the primary, or orthosteric, binding site on the different receptors is very similar.  This similarity is rooted in the fact that the endogenous neurotransmitter acetylcholine affects all nicotinic receptor subtypes.  Therefore, drugs that target the orthosteric site of one subtype, will affect the same site on other subtypes. 

Allosteric modulation is a term that refers to affecting the activity of a receptor by targeting a site other than the orthosteric site.  This is advantageous for nicotinic receptor subtypes because of their diverse structures.  Allosteric modulation aims to find a unique domain on the subtype of interest and to develop drugs that target that binding site.  These drugs will not compete with binding of the primary agonist and are therefore considered to be non-competitive.  This will hopefully bring rise to a drug that can selectively treat various disease states without causing negative side effects. 

Allosteric regulation

In biochemistry, allosteric regulation is the regulation of an enzyme or other protein by binding an effector molecule at the protein's allosteric site (that is, a site other than the protein's active site). Effectors that enhance the protein's activity are referred to as allosteric activators, whereas those that decrease the protein's activity are called allosteric inhibitors. The term allostery comes from the Greek allos (ἄλλος), "other", and stereos (στερεὀς), "solid (object)", in reference to the fact that the regulatory site of an allosteric protein is physically distinct from its active site. Allosteric regulations are a natural example of control loops, such as feedback from downstream products or feedforward from upstream substrates. Long-range allostery is especially important in

cell signaling.[1]

Enzyme stability

The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat.

Enzyme Function

In simple terms, an enzyme functions by binding to one or more of the reactants in a reaction. The reactants that bind to the enzyme are known as the substrates of the enzyme. The exact location on the enzyme where substrate binding takes place is called the active site of the enzyme. The shape of the active site just fits the shape of the substrate, somewhat like a lock fits a key. In this way only the correct substrate binds to the enzyme.

Once the substrate or substrates are bound to the enzyme, the enzyme can promote the desired reaction in some particular way. What that way is depends on the nature of the reaction and the nature of the enzyme. An enzyme may hold two substrate molecules in precisely the orientation needed for the reaction to occur. Or binding to the enzyme may weaken a bond in a substrate molecule that must be broken in the course of the reaction, thus increasing the rate at which the reaction can occur.

An enzyme may also couple two different reactions. Coupling an exothermic reaction with an endothermic one allows the enzyme to use the energy released by the exothermic reaction to drive the endothermic reaction. In fact, a large variety of enzymes couple many different endothermic reactions to the exothermic reaction in which ATP is converted by hydrolysis to ADP. In this way, ATP serves as the molecular fuel that powers most of the energy-requiring processes of living things.

FUNCTIONS OF ENZYMES:

Enzymes serve a wide variety of functions inside living organisms. They are indispensable for 
signal transduction and cell regulation, often via kinases and phosphatases.[77] They also generate movement, with myosin hydrolyzing ATP to generate 
muscle contraction and also moving cargo around the cell as part of the cytoskeleton.[78] Other ATPases in the cell membrane are 
ion pumps involved in 
active transport. Enzymes are also involved in more exotic functions, such asluciferase generating light in fireflies.[79] Viruses can also contain enzymes for infecting cells, such as the HIV integraseand 
reverse transcriptase, or for viral release from cells, like the influenza virus neuraminidase.

An important function of enzymes is in the 
digestive systems of animals. Enzymes such as amylases and proteasesbreak down large molecules (starch or proteins, respectively) into smaller ones, so they can be absorbed by the intestines. Starch molecules, for example, are too large to be absorbed from the intestine, but enzymes hydrolyze the starch chains into smaller molecules such as maltose and eventually glucose, which can then be absorbed. Different enzymes digest different food substances. In ruminants, which have herbivorous diets, microorganisms in the gut produce another enzyme, cellulase, to break down the cellulose cell walls of plant fiber.

Several enzymes can work together in a specific order, creating metabolic pathways. In a metabolic pathway, one enzyme takes the product of another enzyme as a substrate. After the catalytic reaction, the product is then passed on to another enzyme. Sometimes more than one enzyme can catalyze the same reaction in parallel; this can allow more complex regulation: with, for example, a low constant activity provided by one enzyme but an inducible high activity from a second enzyme.

Enzymes determine what steps occur in these pathways. Without enzymes, metabolism would neither progress through the same steps nor be fast enough to serve the needs of the cell. Indeed, a metabolic pathway such as glycolysis could not exist independently of enzymes. Glucose, for example, can react directly with ATP to become phosphorylated at one or more of its carbons. In the absence of enzymes, this occurs so slowly as to be insignificant. However, if hexokinase is added, these slow reactions continue to take place except that phosphorylation at carbon 6 occurs so rapidly that, if the mixture is tested a short time later, glucose-6-phosphate is found to be the only significant product. As a consequence, the network of metabolic pathways within each cell depends on the set of functional enzymes that are present.

ALLOSTERISM

A change in the activity and conformation of an enzyme resulting from the binding of a compound at a site on the enzyme other than the active binding site is called allosterism.

Molecules that increase enzymatic activity (i.e., rates) by binding to sites on the enzyme other than the active site are termed positive allosteric effectors, while molecules that decrease enzymatic activity in a similar fashion are termed negative allosteric effectors.

Allosteric enzymes consist of multiple subunits. Binding of a substrate (or effector) molecule at one subunit induces a conformational change in the subunit. This causes a conformational changes in the other subunits. Thus a significant part of the binding energy of the substrate is used to change the conformation of the protein complex. Binding of a substrate molecule at another binding site does not need alter the structure of the subunit anymore, thus the binding affinity increases. This property changes the curve of activity (v0) versus substrate concentration from a hyperbole (Michaelis-Menten kinetics) into an "S" shape, sigmoidal curve.  

Allosteric sites are attractive therapeutic targets because they can be exploited to achieve modes of selectivity and signaling that are not attainable by orthosteric means. 

e.g: In addition to the orthosteric site, which recognizes endogenous ligands, most G protein-coupled receptors (GPCRs) possess topographically distinct allosteric sites that can be recognized by small molecules and accessory cellular proteins. Ligand binding to allosteric sites promotes a conformational change in the GPCR that can alter orthosteric ligand affinity and/or efficacy.

Allosteric enzymes are enzymes that change their conformational ensemble upon binding of an effector, which results in an apparent change in binding affinity at a different ligand binding site. This "action at a distance" through binding of one ligand affecting the binding of another at a distinctly different site, is the essence of the allosteric concept. Allostery plays a crucial role in many fundamental biological processes, including but not limited to 
cell signaling and the regulation of metabolism. Allosteric enzymes need not be oligomers as previously thought,[1] and in fact many systems have demonstrated allostery within single enzymes.

Allosteric enzymes have two conformations: active (R-state) and less active (T-state)

1. T-state: less active, stabilized by inhibitors

2. R-state: more active, stabilized by substrate and activators

Allosteric enzymes have multiple subunits. Cooperativity results from the R to T transition of subunits and the interaction of these subunits (quaternary structure).

ENZYME STABILITY:

The physical biochemist, on the one hand, would probably discuss protein stability primarily in terms of the thermodynamic stability of a protein that unfolds and refolds rapidly, reversibly, cooperatively, and with a simple, two-state mechanism:

Where Ku, is the equilibrium constant for unfolding.

The easiest proteins in which to study folding and stability are those that exhibit this sort of rapid reversibility. Both experimental design and also theoretical treatment of data are simplified by reversible systems. Thus, it is no surprise that most of the literature reports about stability discuss this type of reversible system. The bulk of this dissertation will also focus on thermodynamic stability.

In these cases, the stability of the protein is simply the difference in Gibbs free energy, G, between the folded and the unfolded states. The only factors affecting stability are the relative free energies of the folded (Gf) and the unfolded (Gu) states. The larger and more positive Gu, the more stable is the protein to denaturation.

The Gibbs free energy, G, is made up the two terms enthalpy (H) and entropy (S), related by the equation:

 

IMMOBILIZATION OF ENZYMES:

Advantages of enzyme immobilization:-

• Multiple or repetitive use of a single batch of enzymes.
• Immobilized enzymes are usually more stable.
• Ability to stop the reaction rapidly by removing the enzyme from the reaction solution.
• Product is not contaminated with the enzyme.
• Easy separation of the enzyme from the product.
• Allows development of a multienzyme reaction system.
• Reduces effluent disposal problems.

 

Disadvantages of enzyme immobilization:-

• It gives rise to an additional bearing on cost.
• It invariably affects the stability and activity of enzymes.
• The technique may not prove to be of any advantage when one of the substrate is found to be insoluble.
• Certain immobilization protocols offer serious problems with respect to the diffusion of the substrate to have an access to the enzyme.