‫ جامعة كركوك‬/ ‫كلية طب االسنان‬
‫محاضرات الكيمياء الحياتية‬
‫ نوال عبدهللا مرتضى‬.‫د‬
‫المرحلة الثانية‬
Enzymes
Chemical Nature of Enzymes
All known enzymes are proteins. They are high molecular weight compounds
(biologic polymers) made up principally of chains of amino acids linked
together by peptide bonds. See Figure 1.
Enzymes catalyze the chemical reactions that make life as we know it
possible. It can be denatured and precipitated with salts, solvents and other
reagents. They have molecular weights ranging from 10,000 to 2,000,000.
Many enzymes require the presence of other compounds ; cofactors - before
their catalytic activity can be exerted. This entire active complex is referred to
as the holoenzyme; i.e., apoenzyme (protein portion) plus the cofactor
(coenzyme, prosthetic group or metal-ion-activator) is called the holoenzyme.
Apoenzyme + Cofactor = Holoenzyme
Apoenzyme
a protein that forms an active enzyme system by combination with a
coenzymeand determines the specificity of this system for a The cofactor may
be:
1- A coenzyme - a non-protein organic substance which is dialyzable, thermo
stable and loosely attached to the protein part.
2- A prosthetic group - an organic substance which is dialyzable and thermo
stable which is firmly attached to the protein or apoenzyme portion.
3- A metal-ion-activator - these include K+, Fe++, Fe+++, Cu++, Co++,
Zn++, Mn++, Mg++, Ca++ and Mo.
A prosthetic group: is a tightly bound, specific non - polypeptide unit
required for the biological function of some proteins. The prosthetic group may
be organic (such as a vitamin, sugar, or lipid) or inorganic (such as a metal
ion), but is not composed of amino acids. Prosthetic groups are bound tightly to
proteins and may even be attached through a covalent bond, as opposed to
coenzymes, which are loosely bound. In enzymes, prosthetic groups are often
involved in the active site, playing an important role in the functions of
enzymes.
Mode of action :
Enzymes enable chemical reactions to occur at cooler temperatures by reducing
the amount of activation energy required to break the bonds of the reactant
molecules. Each enzyme is very selective in the reaction it catalyzes, this
feature is based on the ability of the enzyme to recognize the shape of the
certain reactant molecule - substrate. There is a special region on the enzyme
that has same shape and chemistry as the substrate, so the substrate fits
perfectly into this docking station, the enzyme embraces it slightly and
catalyzes the reaction. When the product is released, the enzyme is ready to
accept another molecule of particular shape. There are two common models of
enzyme action. In the first model, the lock-and-key model, a protein called
an enzyme "the lock" binds with another substance called a substrate "the
key" and causes the lock to break down after forming an enzyme - substrate
complex. The model is so named because substrates are very specific to
individual enzymes. The second model is called the induced-fit model. It is
pretty much the same as the first, except it is understood that the enzyme
undergoes a conformational change before binding with the substrate.
Enzymes are able to facilitate reactions at lower than usual temperatures
because they lower the activation energy requirements of many biological
reactions, acting as organic catalysts. This catalysis often requires "helper"
substances called co-enzymes and cofactors. The rate at which enzymes work
is determined by the type of enzyme, the amount of substrate present, and the
amount of enzyme present. Some molecules can inhibit enzyme function by
imitating the substrate or causing the enzyme to change
shape.
Mechanism of action:
Lock and key" model"
To explain the observed specificity of enzymes, in 1894 Emil Fischer proposed
that both the enzyme and the substrate possess specific complementary
geometric shapes that fit exactly into one another. This is often referred to as
"the lock and key" model. This early model explains enzyme specificity, but
fails to explain the stabilization of the transition state that enzymes achieve.
Induced fit model
In 1958, Daniel Koshland suggested a modification to the lock and key model:
since enzymes are rather flexible structures, the active site is continuously
reshaped by interactions with the substrate as the substrate interacts with the
enzyme. As a result, the substrate does not simply bind to a rigid active site;
the amino acid side - chains that make up the active site are molded into the
precise positions that enable the enzyme to perform its catalytic function. In
some cases, such as glycosidases, the substrate molecule also changes shape
slightly as it enters the active site. The active site continues to change until the
substrate is completely bound, at which point the final shape and charge
distribution is determined. Induced fit may enhance the fidelity of molecular
recognition in the presence of competition and noise via the conformational
proofreading mechanism.
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:
1- Absolute specificity ( absolute, high or substrate specificity):- the
enzyme will catalyze only one reaction and it acts on one substrate e.g.
A- Uricase, which acts only on uric acid.
B- Arginase, which acts only on arginine.
C- Carbonic anhydrase, which acts only on carbonic acid.
D- Lactase, which acts only on lactose.
E- Sucrase, which acts only on sucrose.
F- Maltase, which acts only on maltase.
2- Moderate, structural or group specificity - the enzyme will act only on
molecules that have specific functional groups, such as amino, phosphate and
methyl groups. The enzyme is specific not only to the type of bond but also to
the structure surrounding it.
a- Pepsin: is an endopeptidase that hydrolyzes central peptide bonds in which
the amino group belongs to aromatic amino acids e.g. phenyl alanine, tyrosine
and tryptophan.
b- Trypsin: is an endopeptidase that hydrolyzes central peptide bonds in which
the amino group belongs to basic amino acids e.g. arginine, lysine and histidine.
c- Chymotrypsin: is an endopeptidase that hydrolyzes central peptide
bonds in which the carboxyl group belongs to aromatic amino acids.
d- Aminopeptidase: is an exopeptidase that hydrolyzes peripheral peptide
bond at the amino terminal (end) of polypeptide chain.
e- Carboxypeptidase: is an exopeptidase that hydrolyzes peripheral peptide
bond at the carboxyl terminal of polypeptide chain.
3- Linkage specificity (Relative, low or bond specificity): the enzyme will act
on a particular type of chemical bond regardless of the rest of the molecular
structure. The enzyme acts on substrates that are similar in structure and contain
the same type of bonds e.g.
a- Amylase: acts on α 1-4 glycosidic, bonds in starch, dextrin and glycogen.
b- Lipase
e: hydrolyzes ester bonds in different triglycerides.
4- Stereochemical specificity - the enzyme will act on a particular steric or
optical isomer. In this type of specificity, the enzyme is specific not only to the
substrate but also to its optical configuration e.g.
A- L amino acid oxidase acts only on L amino acids.
B- D amino acid oxidase acts only on D amino acids.
C- α - glycosidase acts only on α – glycosidic bonds, which are present in
starch, dextrin and glycogen.
D- β – glycosidase acts only on β – glycosidic bonds that are present in
cellulose.
Phenylethanolamine N-methyltransferase (PNMT): is an enzyme found in
the adrenal medulla that converts norepinephrine (noradrenaline) to
epinephrine (adrenaline).
1- Oxidoreductases: are classified as EC 1 in the EC number classification of
enzymes. Oxidoreductases can be further classified into 22 subclasses:
EC 1.1 includes oxidoreductases that act on the CH-OH group of donors (alcohol
oxidoreductases).
EC 1.2 includes oxidoreductases that act on the aldehyde or oxo group of donors.
EC 1.3 includes oxidoreductases that act on the CH-CH group of donors (CH-CH
oxidoreductases).
EC 1.4 includes oxidoreductases that act on the CH-NH2 group of donors (Amino acid
oxidoreductases, Monoamine oxidase).
EC 1.5 includes oxidoreductases that act on CH-NH group of donors.
EC 1.6 includes oxidoreductases that act on NADH or NADPH.
EC 1.7 includes oxidoreductases that act on other nitrogenous compounds as donors.
EC 1.8 includes oxidoreductases that act on a sulfur group of donors.
EC 1.9 includes oxidoreductases that act on a heme group of donors.
EC 1.10 includes oxidoreductases that act on diphenols and related substances as donors.
2- Transferases:
EC
Examples
Group(s) transferred
number
EC 2.1
methyltransferase and
single-carbon groups
formyltransferase
EC 2.2
transketolase and transaldolase aldehyde or ketone groups
EC 2.3
acyltransferase
acyl groups or groups that become alkyl
groups during transfer
EC 2.4
glycosyltransferase,
glycosyl groups, as well as hexoses and
hexosyltransferase, and
pentoses
pentosyltransferase
EC 2.5
EC 2.6
riboflavin synthase and
alkyl or aryl groups, other than
chlorophyll synthase
methyl groups
transaminase, and oximino nitrogenous groups
transferase
EC 2.7
phosphotransferase,
phosphorus-containing groups;
polymerase, and kinase
subclasses are based on the acceptor
).(e.g. alcohol, carboxyl, etc
EC 2.8
sulfurtransferase and sulfo sulfur-containing groups
transferase
EC 2.9
selenotransferase
selenium-containing groups
EC 2.10
molybdenumtransferase
molybdenum or tungsten
and tungsten
transferase
3- Hydrolase:
a hydrolase or hydrolytic enzyme is an enzyme that catalyzes the hydrolysis of
a chemical bond. For example, an enzyme that catalyzed the following reaction
is a hydrolase:
A–B + H2O → A–OH + B–H
Hydrolases are classified as EC 3 in the EC number classification of enzymes.
Hydrolases can be further classified into several subclasses, based upon the
bonds they act upon:
EC 3.1: ester bonds (esterases: nucleases, phosphodiesterases, lipase,
phosphatase.
EC 3.2: sugars (DNA glycosylases, glycoside hydrolase).
EC 3.3: ether bonds.
EC 3.4: peptide bonds (Proteases/peptidases).
EC 3.5: carbon-nitrogen bonds, other than peptide bonds.
EC 3.6: acid anhydrides (acid anhydride hydrolases, including helicases and
GTPase).
EC 3.7: carbon-carbon bonds.
EC 3.8: halide bonds.
EC 3.9: phosphorus-nitrogen bonds.
EC 3.10: sulphur-nitrogen bonds.
4- Lyase
a lyase is an enzyme that catalyzes the breaking (an "elimination" reaction) of
various chemical bonds by means other than hydrolysis (a "substitution"
reaction) and oxidation, often forming a new double bond or a new ring
structure. The reverse reaction is also possible (called a "Michael addition”).
For example, an enzyme that catalyzed this reaction would be a lyase:
ATP → cAMP + PPi
Lyases differ from other enzymes in that they require only one substrate for the
reaction in one direction, but two substrates for the reverse reaction.
Lyases are classified as EC 4 in the EC number classification of enzymes.
Lyases can be further classified into seven subclasses:
EC 4.1 includes lyases that cleave carbon-carbon bonds, such as
decarboxylases (EC 4.1.1), aldehyde lyases (EC 4.1.2), oxo acid lyases (EC
)4.1.3), and others (EC 4.1.99).
EC 4.2 includes lyases that cleave carbon-oxygen bonds, such as dehydratases.
EC 4.3 includes lyases that cleave carbon-nitrogen bonds.
EC 4.4 includes lyases that cleave carbon-sulfur bonds.
EC 4.5 includes lyases that cleave carbon-halide bonds.
EC 4.6 includes lyases that cleave phosphorus-oxygen bonds, such as adenylyl
cyclase and guanylyl cyclase.
EC 4.99 includes other lyases, such as ferrochelatase.
5- Isomerase:
Isomerases are a general class of enzymes which convert a molecule from one
isomer to another. Isomerases can either facilitate intramolecular
rearrangements in which bonds are broken and formed. The general form of
such a reaction is as follows:
A–B → B–A
There is only one substrate yielding one product. This product has the same
molecular formula as the substrate but differs in bond connectivity or spatial
arrangements. Isomerases catalyze reactions across many biological processes,
such as in glycolysis and carbohydrate metabolism.
6- Ligase:
a ligase is an enzyme that can catalyze the joining of two large molecules by
forming a new chemical bond, usually with accompanying hydrolysis of a
small pendant chemical group on one of the larger molecules or the enzyme
catalyzing the linking together of two compounds, e.g., enzymes that catalyze
joining of C-O, C-S, C-N, etc. In general, a ligase catalyzes the following
reaction:
Ab + C → A–C + b
or sometimes
Ab + cD → A–D + b + c + d + e + f
where the lowercase letters signify the small, dependent groups. Ligase can
join two complementary fragments of nucleic acid and repair single stranded
breaks that arise in double stranded DNA during replication.
Enzyme kinetics and Km value:
The enzyme (E) and substrate (S) combine with each other to form an unstable
enzyme-substrate complex (ES) for the formation of product (P).
Here k1, k2 and k3 represent the velocity constants for the respective reactions,
as indicated by arrows. Km, the Michaelis - Menten constant (or Brig’s and
Haldane’s constant), is given by the formula:
The following equation is obtained after suitable algebraic manipulation.
where v = Measured velocity, Vmax = Maximum velocity, S = Substrate
concentration,
Km
=
Michaelis
-
Menten
constant.
Km or the Michaelis - Menten constant: is defined as the substrate
concentration (expressed in moles / lit) to produce half - maximum velocity in
an enzyme catalyzed reaction. It indicates that half of the enzyme molecules
(i.e. 50%) are bound with the substrate molecules when the substrate
concentration equals the Km value. Km value is a constant and a characteristic
feature of a given enzyme. It is a representative for measuring the strength of
ES complex. A low Km value indicates a strong affinity between enzyme and
substrate, whereas a high Km value reflects a weak affinity between them. For
Line weaver-Burk double reciprocal plot:
For the determination of Km value, the substrate saturation curve (Fig. 66.2) is
not very accurate since Vmax is approached asymptotically. By taking the
reciprocals of the equation (1), a straight line graphic representation is
obtained. The Line weaver - Burk plot is shown in Fig. 66.3. It is much easier
to calculate the Km from the intercept on x-axis which is -(1/Km). Further, the
double reciprocal plot is useful in understanding the effect of various
inhibitions. majority of enzymes, the Km values are in the range of 10 -5 to 10-2
moles.
Factors Affecting Enzyme Function
1- Effect of concentrations.
a- Enzyme concentration.
b- Substrate concentration.
c- Product Concentration.
2- Temperature.
3- pH.
4- Salinity.
5- Activators.
6- Inhibitors.
1- Effect of concentrations.
a- Enzyme Concentration: Increasing enzyme concentration will increase the
rate of reaction, as more enzymes will be colliding with substrate molecules.
However, this too will only have an effect up to a certain concentration, where
the enzyme concentration is no longer the limiting factor.
- Denaturation: is a permanent structural change in a protein that results in a
loss of its biological properties. In other words, some chemical bonds break,
and the active site is destroyed.
- Human
enzymes: can denature in individuals running a high fever.
- Some arctic: fish have enzymes that denature at 10ºC.
- The enzymes: of some thermophilic bacteria denature at 85ºC.
b- Substrate concentration:
At low substrate concentration the reaction proceeds slowly. This is because
there are not enough substrate molecules to occupy all of the active sites on the
enzyme. As substrate concentration increases, the rate increases because there are
more enzyme substrate complexes formed. At point x, however, increasing the
substrate concentration will have no further effect on the rate of reaction. This is
because all of the enzyme’s active sites are now occupied by substrate molecules
– increasing the substrate concentration further will have no effect, because no
more enzyme substrate complexes can form. The rate of reaction now depends on
the turnover rate of the enzyme, i.e. the number of substrate molecules
transformed
by
one
molecule
of
enzyme
per
second.
c- Effect of Product Concentration:
The accumulation of reaction products generally decreases the enzyme
velocity. For certain’ enzymes, the products combine with the active site of
enzyme and form a loose complex and, thus, inhibit the enzyme activity. In
the living system, this type of inhibition is generally prevented by a quick
removal of products formed.
2- Effect of Temperature: Enzymes have an optimum temperature – this is
the temperature at which they work most rapidly. Below the optimum
temperature, increasing temperature will increase the rate of the reaction. This
is because temperature increases the kinetic energy of the system, effectively
increasing the number of collisions between the substrate and the enzyme’s
active site. Temperatures above the optimum will lead to denaturation. This
occurs because the hydrogen bonds and disulphide bridges which maintain the
shape of the active site are broken. Thus, enzyme substrate complexes can no
longer
be
fo
rmed.
3- Effect of pH: Increase in the hydrogen ion concentration (pH)
considerably influences the enzyme activity and a bell-shaped curve is
normally obtained. Each enzyme has an optimum pH at which the velocity
is maximum. Most of the enzymes of higher organisms show optimum
activity around neutral pH (6-8). There are, however, many exceptions like
pepsin (1-2), acid phosphatase (4-5) and alkaline phosphatase (10-11) for
optimum pH. As with temperature, each enzyme has an optimum pH. If
pH increases or decreases much beyond this optimum, the ionisation of
groups at the active site and on the substrate may change, effectively
slowing or preventing the formation of the enzyme substrate complex. At
extreme pH, the bonds which maintain the tertiary structure – hence the
active site – are disrupted and the enzyme is irreversibly denatured. Since
most human enzymes are intracellular, most have a pH optimum of 7.37.4. However, pepsin, which works in the acidic environment of the
stomach, has an optimum of 2.4.
4- Effect of time:
At the beginning, the rate of reaction increases but by time the rate of
reaction decreases due to :
a- Depletion of substrate.
b- Accumulation of end product.
c- Change in PH of the reaction, becomes different from the optimum PH
of the enzyme.
5- Effect of salinity: Salinity may be important not just in solubility, but in
promoting enzyme activity. Higher salinity may promote binding of a
hydrophobic substrate to an enzyme, or of hydrophobic residues to each other
within the enzyme to ensure optimal folding for enzymatic acti vity.
6- Effect of Activators: Some molecules bind to the enzyme molecule and
consequently increase the reaction rate. These are known as activator
molecules, Like certain inorganic metallic cations ; Mg2+, Mn2+, Zn2+, Ca2+,
Co2+, Cu2+, Na+, K+ etc. for their optimum activity. Rarely, anions are also
needed for enzyme activity e.g. chloride ions (CI–) activate salivary amylase,
calcium ions (Ca+2) activate thrombokinase enzyme (thrombokinase - an
enzyme liberated from blood platelets that converts prothrombin into thrombin
as blood starts to clot). In the conjugated protein enzymes that need coenzyme
the increase in the coenzyme concentration causes an increase in the rate of
enzyme action.
7- Effect of Inhibitors:
Some molecules bind to the enzyme or the substrate or the enzyme - substrate
complex and lower the reaction rate. These are known as inhibitor molecules.
There are two types of inhibitors; 1) Reversible inhibitors and 2) Irreversible
inhibitors.
1) Reversible inhibitors include:
a) competitive inhibition: the substrate and inhibitor cannot bind to the
enzyme at the same time.
b) uncompetitive inhibition: the inhibitor binds only to the substrate-enzyme
complex.
c) non-competitive inhibition: the binding of the inhibitor to the enzyme
reduces its activity but does not affect the binding of substrate.
d) mixed inhibition: the inhibitor can bind to the enzyme at the same time as
the enzyme's substrate.
2) Reversible inhibitors: Traditionally reversible enzyme inhibitors have been
classified as competitive, uncompetitive, or non-competitive, according to their
effects on Km and Vmax. These different effects result from the inhibitor
binding to the enzyme E, to the enzyme–substrate complex ES, or to both,
respectively.
Isoenzymes: (also known as isozymes or more generally as multiple forms of
enzymes) are enzymes that differ in amino acid sequence but catalyze the same
chemical reaction. These enzymes usually display different kinetic parameters
(e.g. different KM values), or different regulatory properties. Isozymes or
Isoenzymes are proteins with different structure which catalyze the same
reaction. Frequently they are oligomers made with different polypeptide
chains, so they usually differ in regulatory mechanisms and in kinetic
characteristic. In biochemistry, isozymes (or isoenzymes) are isoforms (closely
related variants) of enzymes. In many cases, they are coded for by homologous
genes that have diverged over time. Allozymes represent enzymes from
different alleles of the same gene, and isozymes represent enzymes from
different genes that process or catalyse the same reaction, the two words are
usually
used
interchangeably.
Co-enzyme function and important
Coenzymes are a type of cofactor and they are bound to enzyme active sites to
aid with their proper functioning. Coenzymes which are directly involved and
altered in the course of chemical reactions are considered to be a type of
secondary substrate. This is because they are chemically changed as a result of
the reaction unlike enzymes. However unlike the primary substrates,
coenzymes can be used by a number of different enzymes and as such are not
specific. For example hundreds of enzymes are able to use the coenzyme NAD.
The function of coenzymes is to transport groups between enzymes. Chemical
groups include hydride ions which are carried by coenzymes such as NAD,
phosphate groups which are carried by coenzymes such as ATP and acetyl
groups which are carried by coenzymes such as coenzyme A. Coenzymes
which lose or gain these chemical groups in the course of the reaction are often
reformed in the same metabolic pathway. For example NAD+ used in
glycolysis and the citric acid cycle is replaced in the electron transport chain of
respiration.
How are coenzymes made?
Due to the importance of coenzymes in chemical reactions, and due to the fact
that they are used up and chemically altered by reactions, coenzymes must be
continually regenerated. For example, synthesis of B vitamins is a complex,
step wise process because the B vitamins have chiral centres which are
complicated to synthesise. Coenzymes that are produced from B vitamins are
especially important to the proper functioning of enzymes involved with
regulation of metabolism and with the release of energy from food. Important
B vitamins that are used as large components of coenzymes include riboflavin,
niacin, biotin, pantothenic acid, B6, folate and B12. For example riboflavin, or
vitamin B2 is used as a large component of FAD and FADH, and niacin is an
important component of NAD and NADH. However vitamins cannot be made
by the body, but instead they must be consumed in the diet. Therefore vitamins
are essential components of the diet. Although the human body uses more than
its own body weight in ATP, not as much of the vitamin that is used to produce
the coenzyme is needed to be consumed. This is because the body is able to use
the vitamins very instensively through regeneration.