In this essay we will discuss about:- 1. Definition of Enzymes 2. Classification of Enzymes 3. Properties 4. Specificity 5. Preparation and Isolation 6. Recognition 7. Factors Influencing the Action 8. Catalytic Site 9. General Acid or Base Catalysts 10. Mechanism 11. Diagnostic Value.
Contents:
- Essay on the Definition of Enzymes
- Essay on the Classification of Enzymes
- Essay on the Properties of Enzymes
- Essay on the Enzyme Specificity
- Essay on the Preparation and Isolation of Enzymes
- Essay on the Recognition of Enzymes
- Essay on the Factors Influencing the Action of Enzymes
- Essay on the Catalytic Site of Enzyme
- Essay on the Enzymes as General Acid or Base Catalysts
- Essay on the Mechanism of Enzyme Action
- Essay on the Diagnostic Value of Serum Enzymes
Essay # 1. Definition of Enzymes:
Enzymes are soluble, colloidal organic catalysts formed by living cells, specific in action, protein in nature, inactive at 0°C and destroyed by moist heat at 100°C.
Intracellular Enzymes:
Enzymes which are used in the cells which make them are said to be intracellular enzymes. These enzymes correspond to the old “organised ferments”.
Extracellular Enzymes:
Enzymes which are produced by other cells and are secreted to other parts of the body (e.g., digestive juice) are called extracellular enzymes. These enzymes correspond to the old “unorganised ferments”.
Zymase secretion:
An extracellular enzyme which is secreted ready for action is called a zymase secretion.
Example:
Amylase of saliva.
Zymogen secretion:
An enzyme which is secreted in inactive form and ultimately activated by an agent secreted by other cell is said to be zymogen secretion.
Example:
Trypsinogen (in pancreatic juice) activated by enterokinase (in intestinal mucosa) to give active trypsin, prothrombin (in blood) activated by thromboplastin (in tissues) to give active thrombin.
Zymogen secretion is probably a protective mechanism to prevent digestion of cell walls and ducts, since it is most frequently found with protein-splitting enzymes.
Substrate:
The substance on which an enzyme acts is called the substrate.
Example:
Maltose is the substrate on which the enzyme maltase acts to form glucose.
Names:
Except the enzymes ptyalin, pepsin, trypsin and erepsin, enzymes are usually named by adding the suffixase to the main part of the name of the substrate on which they act.
Examples:
Maltase acts on maltose.
Lactase acts on lactose.
Lipases act on lipids.
Carbohydrases act on carbohydrates.
Proteases act on proteins.
Amylases act on starch (Amylum).
But there are many substances which are acted on by some enzymes in different ways. A dipeptide can be attacked by three enzymes. These enzymes are named by their function.
Example:
A dipeptide can be hydrolysed by di-peptidase into amino acids. The free amino group of the amino acid is removed by another enzyme and the free carboxyl group is also removed by another enzyme. So the names of these three enzymes acting on a dipeptide are a di-peptidase, a deaminase and a decarboxylase. Other enzymes are named by their functions only.
Examples:
Transferases, dehydrogenases, hydrolases, oxidases and reductases.
Many enzymes with the same function act on one substance; it is, therefore, better to specify the enzyme by its source.
Example:
Pancreatic amylase, bone phosphatase, liver esterase.
Some enzymes acting on the substrates are freely described by the adjectives.
Example:
Amylolytic, lipolytic, proteolytic.
Catalytic RNAs:
a. Certain ribonucleic acids (RNAs) show highly substrate-specific catalytic activity.
b. These RNAs satisfy the classic criteria for definition as enzymes and are termed ribozymes.
c. Ribozymes catalyse trans-esterification and finally hydrolysis of phosphodiester bonds in RNA molecules. These reactions are enhanced by free OH groups.
d. Ribozymes are involved in the main roles in the intron splicing events essential for the conversion of Pre-mRNA in mature mRNAs.
Essay # 2. Classification of Enzymes:
The 6 major classes of enzymes with some examples are:
a. Oxidoreductases:
Enzymes catalyzing oxidoreductions between two substrates A and B:
Areduced + Boxidized = Aoxidized + Breduced
These enzymes can be grouped in many different ways.
Three main groups can be explained in order to get the simpler expression:
Oxidases:
The enzymes which use oxygen as hydrogen acceptor.
Examples:
Tyrosinase, cytochrome oxidase, uricase.
Anaerobic dehydrogenases:
The enzymes which use some other substance as hydrogen acceptor.
Examples:
Malate dehydrogenase, succinate dehydrogenase, lactate dehydrogenase.
Hydro-peroxidases:
The enzymes which use hydrogen peroxide as substrate.
Examples:
Peroxidase, catalase.
Aerobic dehydrogenases:
The enzymes which use either oxygen or another substance as hydrogen acceptor.
Examples:
D- and L-amino acid oxidases, xanthine oxidase, aldehyde oxidase.
Two other groups are:
Oxygenases:
The enzymes which act on single hydrogen donors with incorporation of oxygen.
Example:
Tryptophan oxygenase.
Hydroxylases:
The enzymes which act on paired donors with incorporation of oxygen into one donor.
Examples:
Steroid hydroxylases, phenylalanine 4-hydroxy- lase.
b. Transferases (Transferring enzymes):
They catalyse the transfer of some group or radical, R, from one molecule A, to another molecule, B:
The group includes:
(i) Transphosphorylases:
Examples:
Hexokinase, phosphoglucomutases, phosphoglycerate kinase:
(ii) Transgtycosidases:
Examples:
Phosphorylase.
(iii) Transaminases:
Examples:
Glutama tepyruvate transaminase, aspartate amino transferase.
(iv) Transatylase:
Examples:
Choline acetyl transferase, acetoacetate transacetylase, amino acid transacetylase:
(iv) Transmethylase:
c. Hydrolases:
Enzymes catalysing hydrolysis of ester, ether, peptide, glycosyl, acid anhydride by the addition of water.
The group includes the extracellular digestive enzymes and many intracellular enzymes:
(i) Enzymes acting on glycosyl compounds, e.g., β-galactosidase. β-D-galactoside + H2O = An alcohol + D- galactose.
(ii) Enzymes acting on peptide bonds, e.g., pepsin, rennin, chymotrypsin.
(iii) Esterases, e.g., lipases, phosphatases, sulphatases.
(iv) Amidases, e.g., urease, arginase, glutaminase.
(v) Hydrolytic deaminases, e.g., guanine deaminase.
Monomeric e.g., Ribonuclease, trypsin carboxypeptidase.
Oligomeric e.g., Acetyl CoA carboxylase, lactate dehydrogenase.
d. Lyases:
Enzymes that catalyze removal of groups from substrates by mechanisms other than hydrolysis leaving double bonds:
(a) Aldehyde-lyases, e.g., aldolase.
Ketose-1-phosphate = dihydroxy-acetone phosphate + an aldehyde.
(b) Carbon-oxygen lyases, e.g., fumerase.
L-malate = Fumerate +H2O.
e. isomerases:
Enzymes catalyzing intercon- version of optical, geometric, or positional isomers.
(i) Racemases and epimerases, e.g., alanine racemase.
L-alanine=D-alanine.
(ii) Cis-trans isomerases, e.g., retinene isomerase.
All transretinene = II-cis-retinene.
(iii) Enzymes catalyzing inter-conversion of aldoses and ketoses, e.g., triose phosphate isomerase.
D-glyceraldehyde-3-phosphate = Dihydroxy-acetone phosphate.
f. Ligases (ligare = to bind):
Enzymes catalyzing the linking together of two compounds couple to the breaking of a pyrophosphate bond in ATP or a similar compound:
(i) Enzymes catalyzing formation of C-S bonds, e.g., succinate thiokinase. GTP+ succinate + CoA = GDP + Pi + succinyl-CoA.
(ii) Enzymes catalyzing formation of C-N bonds, e.g., glutamine synthetase. ATP + L-glutamate + NH4+ = ADP + orthophosphate + L-glutamine.
(iii) Enzymes catalyzing formation of C- C bonds, e.g., Acetyl-CoA carboxylase.
ATP + acetyl-CoA + CO2 = ADP + Pi + malonyl -CoA.
Essay # 3. Properties of Enzymes:
a. Enzymes are proteins. So the amino, carboxyl and sulfhydryl groups of the side chains of amino acids are available for linkage between polypeptide chains.
b. The above-mentioned groups, together with others such as the imidazole ring and the alcoholic group of serine, are also responsible for the union between substrate and enzyme. These groups are termed “active groups” and the region of the protein surface at which they are located is termed “active centre”.
c. The enzyme-substrate complex theory assumes combination of enzymes and substrate and then liberation of enzyme and the reaction product. Thus, Enzyme + Substrate = Enzyme – Substrate Complex.
Enzyme – Substrate Complex = Enzymes + Reaction Product(s).
d. In many cases, a series of reactions may be involved in addition to the above one.
This may be indicated thus:
e. Alkaline phosphatase hydrolyses a number of phosphate esters to produce orthophosphate and an alcohol such as Glucose-6-phosphate → Glucose + Ortho- phosphate.
f. From an energy standpoint enzyme reactions are divided into three classes:
(a) Exergonic reaction means the system undergoes a loss of free energy, e.g., lipase, catalase, urease.
(b) There is little change in free energy and the reactions come to equilibrium; so that product molecules and the substrate molecules are present in constant amounts, e.g., glycogen + inorganic phosphate = glucose-1 -phosphate under the influence of phosphorylase.
(c) Endergonic reaction means the supply of energy in order to proceed the reaction.
g. Many enzymes are conjugated proteins. The prosthetic groups (coenyzmes) are readily detached. These enzymes have special role in metabolic processes. The enzymes catalase and peroxidase have cofactors but no coenzymes.
Essay # 4. Enzyme Specificity:
Enzymes, the organic catalysts, differ from inorganic catalysts in their extraordinary specificity.
A. Reaction Specificity:
a. An enzyme catalyzes only a very few reactions (frequently only one).
Example:
Arginase, catalase, urease only attack arginine, hydrogen peroxide and urea, respectively.
b. Most enzymes can catalyze the same type of reaction (phosphate transfer, oxidation- reduction) with several structurally related substrates.
c. Reactions with alternate substrates take place if they are present in high concentration and also dependent on their affinity to enzyme.
Example:
Histidase acts on histidine and this enzyme acts on tryptophan when it is present in high concentration and it is also dependent on due affinity of that enzyme.
d. A substrate can undergo many reactions but in a reaction specificity one enzyme can catalyse only one of the various reactions.
Example:
In the presence of Acetyl-CoA the enzyme citrate synthetase acts on or catalyse oxaloacetate to citrate.
But when only oxaloacetate is present then another specific enzyme i.e. malate dehydrogenase will catalyze it and forms different products i.e., malate:
B. Optical Specificity:
a. Enzymes show absolute optical specificity for at least a portion of substrate molecule.
Example:
Maltase catalyzes the hydrolysis of α-glycosides but not β-glycosides, while Embden-Meyerhof and direct oxidative pathway catalyze the inter-conversion of D-phosphosugar but not L- phosphosugars. Exceptions, D-amino acid oxidase in kidney acts on L-amino acid.
b. The glycosidases which catalyze the hydrolysis of glycosidic bonds between sugars and alcohols are highly specific for the sugar portion but relatively non-specific for the alcohol portion or aglycone.
c. Many substrates form 3 bonds enzymes. This 3-point attachment can thus confer asymmetry on the symmetric molecule.
d. When enzyme acts on a substrate and produces one kind of isomer which will be the product.
Example:
Succinate dehydrogenase while acts on succinic acid will produce only fumaric acid and not malic acid which is an isomer of it:
C. Bond Specificity:
a. A particular enzyme acts on particular chemical bond.
Example:
Glycosidases on glycosides, alcohol dehydrogenase on alcohol, pepsin and trypsin on peptide bond, esterases on ester linkages.
D. Group Specificity:
a. Particular enzyme acts on particular group.
Example:
Chymotrypsin hydrolyzes peptide bond in which the carboxyl group is contributed by the aromatic amino acids such as tyrosin, tryptophan etc. Carboxy- peptidases split off carboxyl group, amino peptidases split off amino group from the polypeptide chain.
E. Oxidoreductases function in biosynthetic processes (e.g., fatty acid synthesis) tend to use NADPH as reductant but those which function in degradative processes tend to use NAD+ as oxidant.
F. In liver, about 90% of the NADP-specific enzyme occurs extra-mitochondrially. But NAD- specific enzyme of mitochondria is activated by ADP.
Essay # 5. Preparation and Isolation of Enzymes:
a. Tissues are extracted with saline or glycerol. Such extracts do not keep for any length of time.
b. These extracts are carefully dried at low temperature.
c. The dried substance is then grinded to a fine powder which keeps well.
d. Then these powders are extracted with suitable solvents.
e. More active preparations are made by various processes of purification involving dialysis to remove inorganic impurities, adsorption in suitable material or precipitation with suitable reagents. These are done at low temperature.
f. In this way much inactive materials can be removed and the enzymes are only present in tissues in very small amounts. They have the high potency. Over 100 enzymes have been obtained as crystalline proteins which are regarded as the pure enzymes.
Essay # 6. Recognition of Enzymes:
If by adding a neutral solution to some starch at 37°C we obtain sugar, we can conclude that a catalyst is present.
If the neutral solution after boiling fails to produce sugar under similar conditions, we may then conclude that the catalyst is an enzyme and not an inorganic catalyst.
Therefore, in the experiments on enzymes, it is necessary to arrange a control with the boiled enzyme solution along with the test.
Essay # 7. Factors Influencing the Action of Enzymes:
a. Contact between Enzyme and Substrate:
i. Enzyme and substrate must be in contact to form the enzyme-substrate complex.
ii. The enzyme and substrate should be well mixed for efficient reaction to proceed.
iii. Even the insoluble substrates must be made soluble by the help of hydrotropic substances whenever required for good mixing with enzymes.
iv. The digestion of fats which are not in solution is greatly facilitated by fine emulsification of the fat giving a greater area of contact for the lipases.
v. Enzymes which are not soluble but are bound to membranes, their contact with substrate must be maintained by setting up suitable concentration gradient.
b. Concentration of Enzyme and Substrate:
i. The rate of enzyme action is influenced by the concentration of substrate, the amount of enzyme and the time.
ii. In case the concentration of substrate is low in comparison to the concentration of enzyme, not all the active groups of the enzyme molecule will be utilized for the formation of enzyme-substrate complex. The rate of reaction will be low.
iii. In case the concentration of substrate increases, more and more active sites of enzyme molecule will be used for the formation of enzyme-substrate complex. The rate of reaction will increase.
iv. If the substrate concentration is such that all the active groups of the enzyme are utilized, the rate of reaction will be proportional to the substrate concentration.
v. Above this concentration mentioned in (4), no matter how high, the rate is maximum and remains constant unless the substrate inhibits the reaction. If the substrate inhibits, the reaction rate declines.
The effect of substrate concentration on the initial rate of an enzyme reaction is shown above by a graphical representation which is known as a Michaelis curve.
It is seen that at low substrate concentration (up to Y in the Fig. 10.5) the rate increases with increasing substrate concentration. At substrate concentration above Y the rate of the reaction has a constant maximum value, V. This is called the maximum velocity of the reaction.
The kinetic equation can be expressed mathematically by Michaelismenten equation:
Vi is the initial velocity, Vmax the maximum velocity, S the substrate concentration, Km Michaelis constant.
The substrate concentration that produces half-maximal velocity is called Michaelis constant or Km value:
When the enzyme concentration is constant, the rate of velocity of enzyme action increases as the concentration of substrate in a hyperbolic manner.
In the following reaction, a typical enzyme action is considered
The rate of formation of ES is proportional to the concentration of uncombined enzyme i.e. [E] – [ES] and concentration of substrate [S].
At maximum velocity all the enzymes are present in combination with substrates.
c. Temperature:
(i) The rise in temperature accelerates an enzyme reaction but at the same time causes inactivation of the enzymes due to denaturation of the protein.
(ii) At a certain temperature known as the optimum temperature the activity is maximum.
(iii) The optimum temperature for man is in the region of 37°C and for most animals in the region of 40°C. The optimum temperature of plant urease is 60°C. The optimum temperature for enzymes from microorganisms adapted to growth in natural hot springs is close to the boiling point of water.
(iv) If the temperature is lowered, the rate of an enzyme reaction is diminished. At the temperature 0°C, most enzymes are practically inactive.
Q10 or temperature coefficient:
i. The rate of an enzyme-catalyzed reaction increases depending over a strictly limited range of temperature owing to increased kinetic energy of the reacting molecules. The kinetic energy of the enzyme exceeds the energy barrier for breaking the weak hydrogen and hydrophobic bonds that maintain its secondary-tertiary structure.
ii. The factor responsible for the increase of the rate of a biologic process for a 10°C temperature rise is the Q10 or temperature coefficient.
iii. The rate of contraction of an excised heart approximately doubles with a 10°C rise in temperature (Q10 = 2).
iv. The change in the rates of many enzyme- catalyzed reactions being accompanied by a rise or fall in body temperature constitutes an essential survival feature for life forms such as lizards which do not maintain a constant body temperature.
v. Homeothermic organisms such as humans can tolerate only a strictly limited change in body temperature and the alterations in reaction rate owing to a change in temperature are of little physiologic significance but are significant in fever or hypothermia. The enzymes in case of homeothermic organisms are able to lower the energy barriers for reactions and to elevate local reactant concentrations.
d. Hydrogen ion Concentration or pH:
(i) A small change in pH may inhibit enzyme activity.
(ii) Pepsin works only in acid medium and is inactivated by making the medium alkaline.
(iii) Trypsin rather works in alkaline solution and cannot digest protein in acid solution.
(i) The maximum activity of the enzyme is at the optimum pH. This value is generally between 5.0 and 9.0.
(v) (i) At the optimum pH, the enzyme (E–) will react with substrate (S+) as:
E− + S+ → ES
At low pH values E will be protonated and lose its negative charge:
E− + H+ → E
At the very high pH values, S+ will lose its positive charge:
S+ →S + H+
e. Oxidation:
(i) The sulfhydryl (SH) groups of many enzymes are essential for enzyme activity.
(ii) Oxidation of these (SH) groups by many oxidizing agents including the oxygen of air forms the disulfide (S-S) linkages and results in loss of enzyme activity.
(iii) Full activity may be regained by reduced sulfhydryl compounds such as cysteine or glutathione (R-SH).
f. Radiation:
(i) Enzymes are highly sensitive to short wave length (high energy) radiation such as X, β- or γ- rays.
(ii) High energy radiation forms peroxides which causes oxidation of the enzyme resulting in loss in enzyme activity.
(iii) The enzyme activity is lost by irradiation which may support indirect effects on the DNA of genes.
g. Coenzymes and Activators:
(i) Enzymes (excepting the enzymes of G.I.T.) work efficiently in presence of some other substances. These substances may be organic and inorganic known as coenzymes and activators respectively.
(ii) In absence of coenzymes and activators the enzymes may be inactive or sluggish.
(iii) The activators (Cl−, Mg++, Ca++, Mn++ etc.) may take part in the formation of enzyme- substrate complex.
(iv) Mn++ in the action of some peptidases may prevent the inactivation of the enzyme by inhibitors.
(v) Some enzymes which are activators are called kinases, e.g., enterokinase converts trypsinogen to trypsin by removing hexapeptide from trypsinogen.
h. Inhibiting Agents:
(i) Many enzymes are inhibited by the salts of mercury, silver, gold and salts of heavy metals or fluorides.
(ii) Oxidases are generally inhibited by cyanides.
(iii) Certain preservatives such as chloroform, glycerol and thymol inhibit some enzymes.
(iv) Toluene has no action on enzymes but is the best preservative for enzyme solutions.
(v) Formaldehyde destroys enzymes.
(vi) The inhibitors present in an enzyme solution occupy the active sites of the enzyme leaving free active sites for substrates to combine.
For example, many enzymes (known as “-SH enzymes”) depend for their activity on the presence of free -SH groups. They can be inactivated by mercuric chloride which reacts with free -SH groups, thus:
This type of inhibition is non-specific.
(vii) (i) The specific inhibitor which is structurally so similar to the substrate molecule that the inhibitor can combine with the enzyme in place of the substrate decreasing the enzyme- substrate union. The inhibitor competes with the substrate for the active group. This is known as “Competitive inhibition”.
The followings are the examples of competitive inhibition:
(a) Succinic acid (HOOC.CH2.CH2 COOH) is converted to fumaric acid when it combines with succinate dehydrogenase; the fumaric acid is released from the enzyme complex leaving the enzyme free to unite with more succinic acid:
Malonic acid (HOOC. CH2. COOH) can also combine with the enzyme but does not undergo any change and is not readily released from the enzyme complex. So the enzyme is not available for union with succinic acid. In such a way malonic acid inhibits succinate dehydrogenase.
(b) Sulphanilamide competes P-amino benzoic acid:
P-amino-benzoic acid is essential for the synthesis of folic acid by the enzyme action of the enzyme in the bacteria. The union of sulphanilamide with the enzyme prevents the union of the enzyme with the P-amino-benzoic acid. Synthesis of folic acid is thus completely prevented.
(ii) Competitive inhibition is reversible.
(iii) Vmax is same.
(iv) Km is increased.
(v) Inhibitor cannot bind with ES complex.
(vi) Complex is EI.
(vii) Michaelis-Menten equation is changed to
(h) (i) Some inhibitors are attached not to the active group but to some other group of enzymes. Under such conditions the activity of the unoccupied active group is affected; so that union with the substrate occurs less readily or not at all. This type of inhibition is called “Non-competitive inhibition”.
(ii) Non-competitive inhibition is reversible or irreversible.
(iii) Vmax is lowered.
(iv) Km is unaltered.
(v) Inhibitor can bind with ES complex.
(vi) Complex is E-S-I or E-E
(vii) Michaelis-Menten equation is changed to
Enzymes which show “allosteric” inhibition have two sites:
(i) Isosteric site,
(ii) Allosteric site.
(i) Isosteric site:
It can bind the substrate or other molecules structurally similar to it.
(ii) Allosteric site:
It can bind other substances.
When the allosteric site binds a molecule, the isosteric site is changed in shape so that the substrate can no longer be bound and hence the enzyme is inhibited.
This is superficially similar to non-competitive inhibition. But it is more complex because the substrate can also affect the shape of the allosteric site and for this reason allosteric inhibition is regarded as a distinct process.
Allosteric inhibitors change the substrate saturation curve to the right. The presence of activators shifts the curve to the left.
Aspartate carbamoylase and phosphofructokinases, the allosteric inhibitor, lower the substrate affinity to increase the Km of the enzyme, but the Vmax is unchanged.
Acetyl-CoA Carboxylase, the allosteric inhibitor, reduces the maximum velocity but no change in Km or substrate affinity.
Feedback allosteric inhibition results when the enzyme is allosterically inhibited by the final product e.g., CTP. The pyrimidine nucleotide inhibits aspartate transcarbamoylase allosterically in the synthesis of Pyrimidine.
The feed-forward allosteric activation of an enzyme for a subsequent step of its metabolism is regulated by a metabolite, e.g., Fructose-1, 6-diphosphate allosterically activates pyruvate kinase catalyzing subsequent step.
Two allosteric enzymes catalyzing reverse reactions are influenced by an allosteric effector, e.g., AMP allosterically activates phosphofructokinase and allosterically inhibits fructose di-phosphatase.
Examples of allosteric modulation:
Both the allosteric site and active site are located on different subunits of oligomeric enzymes. Alternation in the enzyme substrate interaction owing to the allosteric effects of regulatory molecules other than the substrate are said to be heterotopic allosteric modulations.
The positive and negative arrangements with the substrate are exhibited by allosteric activators and inhibitors, respectively. Binding of substrate to one promoter accelerates the binding of the same to another promoter on the same enzyme molecule.
Homo-tropic allosteric effect results in the binding of a substrate enhancing the interaction between the allosteric enzyme and more molecules of the same substrate.
(i) Reversal of inhibition can be brought about by increasing the amount of substrate relative to inhibitor.
(ii) Inhibition may also be reversed by removal of the inhibitor by treatment with hydrogen sulphide (H2S):
i. Anti-Enzymes:
i. If certain enzymes are repeatedly injected into an animal, a substance will be produced in the animal’s serum which can prevent the normal action of the enzymes injected. This substance is called an anti- enzyme.
Examples:
Anti-pepsin, antitrypsin, anti-rennin, anti-urease.
ii. Some people believe that the mucus membrane contains suitable antienzymes for which the alimentary canal is not digested by its own secretions.
Essay # 8. Catalytic Site of Enzyme:
Some restricted region of the enzyme which was concerned with the process of catalysis was termed the active site. In the beginning, the biochemists were puzzled why enzymes were so large, when only a portion of their structure was involved in substrate binding and catalysis. Today, it is clear from 3-dimensional models of enzymes that a far greater portion of the protein interacts with the substrate.
Rigid Model of the Catalytic Site:
The lock and key or rigid template model (see Fig. 10.18) is still useful for understanding certain properties of enzymes:
Flexible Model of the Catalytic Site:
In the Fischer model, the catalytic is presumed to be pre-shaped to fit the substrate. In the induced fit model, the substrate induces a conformational change in the enzyme.
This aligns amino acid residues or other groups on the enzyme in the correct spatial orientation for substrate binding, catalysis, or both.
At the same time, other amino acid residues may be buried in the interior of the molecule. Hydrophobic groups (hatched portion) and charged groups (dots) both are involved in substrate binding.
A phosphoserine (-P) and the -SH of a cysteine are involved in catalysis. Other residues involved in neither process are represented by the side chains of lysine and methionine.
In the absence of substrate, the catalytic and the substrate-binding groups are several bond distances removed from one another. Approach of the substrate induces a conformational change in the enzyme. At the same time, the spatial orientations of other regions are also altered, the lysine and methionine are now closer together.
In the representation of a catalytic site shown in Fig. 10.20, several regions of a polypeptide chain contribute amino acid residues to the site.
Three types of amino acid residues are distinguished in enzymes:
a. Contact residue:
An amino acid residue with one bond distance (0.2 nm) of the substrate.
b. Specificity residue:
An amino residue is involved in substrate binding as well as in catalytic process.
c. Catalytic residue:
An amino acid residue is directly involved in covalent bond changes during enzyme action.
Substrate analogs may cause some of the conformational changes (Fig. 10.21). On attachment of the true substrate (A), all groups (shown as closed circles) are brought into correct alignment. Attachment of a substrate analog that is too “bulky” (Fig. 10.21B) or too “slim” (Fig. 10.21C) induces incorrect alignment.
The exact sequence of events in a substrate induced conformational change remains to be established. Several possibilities stand (Fig. 10.22).
It is still difficult to decide exactly which residues constitute the catalytic site even after knowing the complete primary structure of an enzyme.
Modifiers of Enzyme Activity:
Like all mammalian proteins, enzymes are degraded to amino acids. Although this mechanism reduces enzyme concentration and, hence, catalytic activity, still they are slow, wasteful of carbon and energy and, rather, they are like turning out a light by smashing the bulb, then inserting a new one when light is needed.
The catalytic activity of certain key enzymes can be reversibly decreased or increased by small molecules. Small molecule modifiers which decrease catalytic activity are termed negative modifiers and those which increase or stimulate activity are called positive modifiers.
Essay # 9. Enzymes as General Acid or Base Catalysts:
Reactions whose rates vary as regards to changes in H+ or H3O+ concentration but are independent of the concentrations of other acids or bases present in the solution are said to be specific acid or specific base catalysis.
Reactions whose rates are responsive to all acids or bases present in solution are said to be general acid or general base catalysis. Mutarotation of glucose is one reaction subject to general acid-base catalysis.
Role of Metal Ions:
More than 25 per cent of the enzymes contain tightly-bound metal ions for their activity. The functions of these metal ions may be studied by X-ray crystallography, nuclear magnetic resonance (NMR) and electron spin resonance (ESR).
Metal loenzymes and metal-activated enzymes:
A definite quantity of functional metal ion is present in metallo-enzymes and that metal ion is also retained throughout purification. Metal-activated enzymes bind metals less tightly but require added metals. The mechanism of action in both cases appears to be similar.
Ternary Enzyme-Metal-Substrate Complexes:
Four schemes (shown below) are possible for ternary (3-component) complexes of the catalytic site (Enz), a metal ion (M), and substrate (S) that exhibit 1:1:1 stoichiometry.
The above four schemes are possible for metal- activated enzymes. Metalloenzymes cannot form the Enz-S-M complex, because they retain the metal throughout purification.
Three stages can be stated:
a. Most kinases form substrate-bridge complexes of the type Enz-nucleotide-M.
b. Phosphotransferases using pyruvate or phosphoenolpyruvate as substrate can form metal-bridge complexes.
c. A given enzyme may form one type of bridge complex with one substrate and a different type with the other.
Enzyme-Bridge Complexes (M-Enz-S):
The metals in enzyme-bridge complexes perform structural roles maintaining an active conformation (e.g., glutamine synthase) or form a metal-bridge to a substrate (e.g., pyruvate kinase).
The metal ion in pyruvate kinase also holds one substrate (ATP) to activate it:
Substrate-Bridge Complexes (Enz-S-M): This complex with ATP involves the displacement of water from the coordination sphere of the metal by ATP.
Metal-Bridge Complexes
Activation by metal ions for many peptidases is a slow process which requires many hours. The slow reaction is probably conformational re-arrangement of the binary Enz-M complex to an active conformation.
However, for metalloenzymes, the ternary metal-bridge complex is formed by combination of the substrate (S) with the binary Enz-M complex.
Role of Metal Ions in Catalysis:
Role of Metal Ions to Catalysts:
Metal ions may take part in each of the four mechanisms by which enzymes can accelerate the rates of chemical reactions:
a. General acid-base catalysis.
b. Covalent catalysis.
c. Approximation of reactants.
d. Induction of strain in the enzyme or substrate.
Metal ions like protons can share an electron pair forming a sigma bond. They may also be considered “super acids” and may form pi bonds. Unlike protons, metals can serve as 3-dimensional templates for orientation of basic groups on the enzyme or substrate.
Metal ions can also accept electrons via sigma or pi bonds to activate electrophiles or nucleophiles (general acid-base catalysis). They can also activate nucleophiles or act as nucleophiles themselves by donating electrons.
The coordination sphere of a metal may bring together enzyme and substrate or form chelate-producing distortion in either the enzyme or substrate (strain). A metal ion may also “mask” a nucleophile and thus prevent side reaction.
Essay # 10. Mechanism of Enzyme Action:
a. Fisher in 1894 first proposed that enzyme is like key which fits into lock.
b. In 1913, Michaelis and Menton proposed that enzyme (E) combines with the substrate (s) to form enzyme-substrate complex (ES).
This complex finally gives rise to the reaction product (s) (P) and the enzymes becomes free for further reaction:
During the complex formation the substrate molecules are attached to the specific groups on the enzyme molecules.
These specific groups are said to be ‘active groups’ and the regions on which these groups are located are said to be “active sites”. The active groups are sulfhydryl (-SH) group of cysteine, phenolic group of tyrosine (as in pepsin), alcoholic group of serine (as in trypsin and chymotrypsin) and imidazole group of histidine.
c. In 1963. Koshland proposed “induced fit mechanism’ for mode of enzyme action. According to this, the substrate fits the active sites effectively by inducing a configurational change in the enzyme.
Thus, the complexes are formed by multiple bonding (covalent, hydrogen or electrostatic). The functional groups of the active sites are arranged in a definite spatial configuration for which d-or l – isomer of a substance can act as a substrate for an enzyme.
Many Hormones act through Allosteric Second Messengers:
a. Many hormones bind to their cell surface receptors and many nerve impulses and bring about changes in the rate of enzyme-catalyzed reactions inside target cells by the release or synthesis of specialized allosteric effectors called Second messengers.
b. 3′, 5′-cAMP is the best-known second messenger. This is synthesized from ATP by the enzyme adenylyl cyclase by the effect of the hormone epinephrine.
c. Calcium ion, stored inside the sarcoplamic reticulum of muscle cells, is released into the cytoplasm by the effect of depolarization of membrane resulting from a nerve impulse opening the membrane channel.
Calcium ion binds to and activates enzymes which are involved in the regulation of contraction and the mobilization of stored glucose from glycogen. The glucose then supplies the increased energy demands of muscle contraction.
d. Another second messengers are 3′, 5′- cGMP and polyphosphoinositols which are produced by the hydrolysis of inositol phospholipids by hormone-regulated phospholipases.
Pro-enzymes Facilitate Rapid Mobilization of an Activity in Response to Physiologic Demand:
a. Certain proteins are required essentially at all times.
b. Others (e.g., the enzymes of blood clot formation and dissolution) are required only intermittently. When these enzymes are required, they are needed rapidly.
c. Some physiologic processes such as digestion are intermittent but fairly regular.
d. Others such as blood clot formation, dissolution, and tissue repair need to be brought “on line” in response to physiologic or pathophysiologic need.
e. The synthesis of proteases as inactive precursor proteins protect the tissue of origin (e.g., the pancreas) form auto-digestion which can occur in pancreatitis.
f. De novo synthesis of the required proteins is not sufficiently rapid to respond to pathophysiologic demand such as the loss of blood.
g. The secretion process is slow relative to the physiologic demand.
Essay # 11. Diagnostic Value of Serum Enzymes:
Very small amounts of enzymes which are involved in the reactions in the tissues are present in blood under normal conditions. The concentrations of these enzymes are significantly increased due to their more liberation by the affected tissues to the blood stream under certain clinical conditions.
Increase in the enzyme activities in cerebrospinal fluid is not reflected in the blood. Changes may occur in the cerebrospinal fluid enzymes in certain diseases of the central nervous system.Lactate dehydrogenase activity in cerebrospinal fluid is increased frequently in meningitis, cerebral thrombosis and hemorrhage.
Glucose phosphate isomerase concentration in the cerebrospinal fluid is also elevated in malignant tumors of the brain and frequently in meningitis and cerebral thrombosis. The determination of the activity of the following enzymes shown in the list can give valuable confirmatory or suggestive diagnostic evidence to the physicians.