ENZYMES
Enzymes are protein polymers that posses the ability to specifically recognize biological
molecules, bind to them and catalyse a chemical reaction.. Enzymes allow many
chemical reactions to occur within the homeostasis constraints of a living system.
Enzymes function as organic catalysts. A catalyst is a chemical involved in, but not
changed by, a chemical reaction. Many enzymes function by lowering the activation
energy of reactions. By bringing the reactants closer together, chemical bonds may be
weakened and reactions will proceed faster than without the catalyst.
Enzymes can act rapidly, as in the case of carbonic anhydrase (enzymes typically end in
the -ase suffix), which causes the chemicals to react 107 times faster than without the
enzyme present. Carbonic anhydrase speeds up the transfer of carbon dioxide from cells
to the blood. There are over 2000 known enzymes, each of which is involved with one
specific chemical reaction. Enzymes are substrate specific. The enzyme peptidase (which
breaks peptide bonds in proteins) will not work on starch (which is broken down by
human-produced amylase in the mouth).
Nomenclature
Except for some of the originally studied enzymes such as pepsin, rennin, and trypsin,
most enzyme names end in "ase". The International Union of Biochemistry (I.U.B.)
initiated standards of enzyme nomenclature which recommend that enzyme names
indicate both the substrate acted upon and the type of reaction catalyzed. Under this
system, the enzyme uricase is called urate: O2 oxidoreductase, while the enzyme glutamic
oxaloacetic transaminase (GOT) is called L-aspartate: 2-oxoglutarate aminotransferase.
Enzymes can be classified by the kind of chemical reaction catalyzed.
1. Addition or removal of water
1. Hydrolases - these include esterases, carbohydrases, nucleases,
deaminases, amidases, and proteases
2. Hydrases such as fumarase, enolase, aconitase and carbonic anhydrase
2. Transfer of electrons
1. Oxidases
2. Dehydrogenases
3. Transfer of a radical
1. Transglycosidases - of monosaccharides
2. Transphosphorylases and phosphomutases - of a phosphate group
3. Transaminases - of amino group
4. Transmethylases - of a methyl group
5. Transacetylases - of an acetyl group
4. Splitting or forming a C-C bond
1. Desmolases
5. Changing geometry or structure of a molecule
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1. Isomerases
6. Joining two molecules through hydrolysis of pyrophosphate bond in ATP or other
tri-phosphate
1. Ligases
Mechanism
The catalytic action of enzymes is dependent on;
a) Substrate type; Enzymes select the reaction to catalyse. Enzymes are proteins. The
shape of the protein determines the functioning of the enzyme. The lock and key ‘ theory
explains that each enzyme is specific for one and only one substrate, thus preserving
order in living cells. They have active sites, where the reacting molecules fit. The
arrangement of molecules on the enzyme produces the active site within which the
specific substrate(s) will "fit". It recognizes, confines and orients the substrate in a
particular direction. To be effective, some enzymes esp. oxidizing enzymes require coenzymes (conjugated protein) or activators/ cofactors in form of metallic / non-metallic
ions such as potassium or chloride ions.
b) Temperature: Increases in temperature will speed up the rate of nonenzyme mediated
reactions, and so temperature increase speeds up enzyme-mediated reactions, but only to
a point. When heated too much, enzymes (since they are proteins dependent on their
shape) become denatured. When the temperature drops, the enzyme may regain its shape.
Thermolabile enzymes, work better (or work at all) at lower temperatures.
c) Concentration of substrate and product also control the rate of reaction, providing a
biofeedback mechanism.
d) Changes in pH will also denature the enzyme by changing the shape of the enzyme.
Enzymes are also adapted to operate at a specific pH or pH range. Most enzymes are
effective in a nearly neutral environment hence become inactivated if the medium
becomes strongly acid or alkaline. Some however like pepsin can operate in an acid
medium and becomes inactivated when the food passes through the small intestines
which has a neutral environment.
e) Allosteric Interactions may allow an enzyme to be temporarily inactivated. Binding
of an allosteric effector changes the shape of the enzyme, inactivating it while the
effector is still bound. Such a mechanism is commonly employed in feedback inhibition.
Often one of the products, either an end or near-end product act as an allosteric effector,
blocking or shunting the pathway.
f) Competitive Inhibition works by the competition of the regulatory compound and
substrate for the binding site. They have a close natural resemblance to the natural
substrate and compete with the substrate for the active site of the enzyme. If enough
regulatory compound molecules bind to enough enzymes, the pathway is shut down or at
least slowed down.
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g) Noncompetitive Inhibition occurs when the inhibitory chemical, which does not have
to resemble the substrate, binds to the enzyme other than at the active site. Lead binds to
SH groups in this fashion. The degree of inhibition is affected only by inhibitor
concentration. Irreversible Inhibition occurs when the chemical either permanently binds
to or massively denatures the enzyme so that the tertiary structure cannot be restored.
Nerve gas permanently blocks pathways involved in nerve message transmission,
resulting in death. Penicillin, the first of the "wonder drug" antibiotics, permanently
blocks the pathways certain bacteria use to assemble their cell wall components.
Specificity of Enzymes
One of the properties of enzymes that makes them so important as diagnostic and
research tools is the specificity they exhibit relative to the reactions they catalyze. A few
enzymes exhibit absolute specificity; that is, they will catalyze only one particular
reaction. Other enzymes will be specific for a particular type of chemical bond or
functional group. In general, there are four distinct types of specificity:
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Absolute specificity - the enzyme will catalyze only one reaction.
Group specificity - the enzyme will act only on molecules that have specific
functional groups, such as amino, phosphate and methyl groups.
Linkage specificity - the enzyme will act on a particular type of chemical bond
regardless of the rest of the molecular structure.
Stereochemical specificity - the enzyme will act on a particular steric or optical
isomer.
Though enzymes exhibit great degrees of specificity, cofactors may serve many
apoenzymes. For example, nicotinamide adenine dinucleotide (NAD) is a coenzyme for a
great number of dehydrogenase reactions in which it acts as a hydrogen acceptor. Among
them are the alcohol dehydrogenase, malate dehydrogenase and lactate dehydrogenase
reactions.
Specificity is important in food processing where only a single component may be
modified in the process. The accuracy of enzymatic methods in food analysis depends on
specificity.
Enzyme Kinetics
Energy Levels
Chemists have known for almost a century that for most chemical reactions to proceed,
some form of energy is needed. They have termed this quantity of energy, "the energy of
activation." It is the magnitude of the activation energy which determines just how fast
the reaction will proceed. It is believed that enzymes lower the activation energy for the
reaction they are catalyzing.
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The enzyme is thought to reduce the "path" of the reaction. This shortened path would
require less energy for each molecule of substrate converted to product. Given a total
amount of available energy, more molecules of substrate would be converted when the
enzyme is present (the shortened "path") than when it is absent. Hence, the reaction is
said to go faster in a given period of time.
Basic Enzyme Reactions
Enzymes are catalysts and increase the speed of a chemical reaction without themselves
undergoing any permanent chemical change. They are neither used up in the reaction nor
do they appear as reaction products. It is believed that for this to occur the following
should take place:
- The enzyme must form a temporary association with the substance or substances
whose reaction rate it affects (substrate)
- The association between enzyme and substrate is thought to form a close physical
association between the molecules and is called the enzyme- substrate complex.
- While the enzyme-substrate complex is formed. Enzyme action takes place
- Upon completion of the reaction, the enzyme and products separate. The enzyme
molecule is now available to form additional complexes. The reaction is
summarized by Michaelis- Menten equation.
E+S
-
K1
ES
K-1
K2
E+P
The substrate S combines with the enzyme E, to form an intermediate complex
ES which then breaks to product P and free enzyme. The equilibrium constant
for the formation of the complex is called Michaelis constant Km
(defined as K2 + K-1/ K1)
The rate of reaction is a function of the enzyme and substrate concentration and is
affected by the presence of activators or inhibitors.
Enzyme inhibition
-
Enzyme inhibitors may be reversible or not reversible. In reversible inhibition, the
enzyme activity is restored upon removal of the inhibitor.
Irreversible inhibition is progressive and becomes complete when all the enzyme
is combined with the inhibitor. The enzyme is not reactivated even after removal
of the inhibitor. Heavy metals eg. mercury, are examples of irreversible inhibitors.
Enzyme activity in food processing
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Since enzymes have the ability to react specifically with individual components of
a mixture, they can be used in analysis hence the term “Enzymatic Analysis”.
In this technique, the presence of contaminants in a sample may partially or
totally inhibit the enzyme - catalysed reactions, thus creating a problem. Results
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may also be affected by heavy metals and oxidants. Compounds structurally
similar to the enzyme but without biological activity may be competitive
inhibitors. Hence for good results, the preparation must be pure. Immobilized
enzymes can be used as analytical reagents to reduce the cost of purifying
preparations.
Immobilized enzymes are physically or chemically restricted in movement so
that it can be physically reclaimed from the reaction medium. They have nearly
the same activity as free enzymes.
They are highly stable, reusable, can be easily removed from the reaction without
contamination. They enable controlled product formation, greater variety of
engineering designs for continuous processes, Long half life with predictable
decay rate. Immobilized enzymes can be prepared in physical and chemical ways;
a)
b)
c)
d)
Adsorption
physical
Entrapment with a gel matrix
physical
Covalent cross linking to itself or to a second protein - chemical
Covalent attachment to an insoluble carrier such as a polysaccharide –
chemical
Chemical methods are irreversible because the original enzyme cannot be regenerated or
recovered. Physical methods do not involve covalent bonding, hence, are reversible. It is
important to note that immobilization can affect enzyme activity, stability, specificity, pH
optimum as well as Michaellis - Menten constant.
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Immobilized enzymes are used together with enzyme electrodes in the following
procedures (electrodes are based on consumed or produced hydrogen peroxide)
a) determination of sucrose in food products
b) determination of starch and maltose
c) simultaneous determination of sucrose and glucose, lactose and glucose,
starch and glucose
d) assay of aspartame
Determining enzyme activity in food processing.
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Proteinaise activity can be determined through changes in the modified substrate
ie. determination of rheological parameters (viscosity, dough consistency,
coagulation, solubility) or through an assay of fractions split off from a substrate
where carboxylic or amino groups can be titrated or free amino acids can be
determined by specific or non – specific (ninhydrin) reactions. Soluble protein
not precipitated are measured before and after proteinaise action and the
difference gives an estimate of enzymatically modified substrate.
Activity of peptidases is assayed by microtitration or colorimetry of amino acids.
Urease activity is used as an index of heat treatment of soy flour toasted to
improve palatability and nutritional value.
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Starch liquefying - amylase are determined by viscometric, dextrinogenic, or
colorimetric assays. Determination of - amylase activity is useful in detecting
sprouted grain and in control of malted production and supplementation.
Phosphatases in milk are assayed to determine efficiency of pasteurization of milk
and milk products since they are activated within temperature range used in milk
pasteurization.
To determine soundness of stored grain, fatty acidity is used as an index.
The efficiency of blanching fruits and vegetables is determined by testing
inactivation of peroxidase.
In commercial food processing, enzymes are also used to alleviate a processing
difficulty or improve the product. The following are some of the applications.( See
attached handout).
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