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BCH 234

INTRODUCTORY LECTURE NOTES ON ENZYMES BY DR. A.N. OKPOGBA

Enzymes and Life Processes

The living cell is the site of tremendous biochemical activity called metabolism. This is the process of
chemical and physical change which goes on continually in the living organism such as build-up of
new tissue, replacement of old tissue, conversion of food to energy, disposal of waste materials,
reproduction - all the activities that we characterize as "life."

This building up and tearing down takes place in the face of an apparent paradox. The greatest
majority of these biochemical reactions do not take place spontaneously. The phenomenon of catalysis
makes possible biochemical reactions necessary for all life processes. Catalysis is defined as the
acceleration of a chemical reaction by some substance which itself undergoes no permanent chemical
change. The catalysts of biochemical reactions are called enzymes and are responsible for bringing
about almost all of the chemical reactions in living organisms. Without enzymes, these reactions take
place at a rate far too slow for the pace of metabolism.

The oxidation of a fatty acid to carbon dioxide and water requires extremes of pH, high temperatures
and corrosive chemicals but in the body, such a reaction takes place smoothly and rapidly within a
narrow range of pH and temperature. In the laboratory, the average protein must be boiled for about 24
hours in a 20% HCl solution to achieve a complete breakdown but in the body, the breakdown takes
place in four hours or less under conditions of mild physiological temperature and pH.

It is through attempts at understanding more about enzyme catalysts - what they are, what they do, and
how they do it - that many advances in medicine and the life sciences have been brought about.

History of Enzymes

Enzymes are generally proteins that are synthesized in a living cell and catalyze or speed up a
thermodynamically possible reaction so that the rate of the reaction is compatible with the
biochemical process essential for the maintenance of a cell. They are biological catalysts as against
inorganic catalysts. Enzymes provide speed, specificity and regulate control to the reactions of the
body. Enzymes increase the rate at which reactions approach equilibrium but do not affect it and
remain unchanged after the reaction. Although enzymes are subject to the same laws of nature that
govern the behavior of other substances, they differ from ordinary chemical catalysts in several
respects:

1. Enzymes provide higher reaction rates up to 10 6 to 1012 than uncatalysed reactions and at least
several orders of magnitude greater than those of the corresponding chemically catalysed
reactions.
2. Enzymes operate under milder conditions of temperature, at least below 100 0C, mild conditions of
atmospheric pressure and nearly neutral pHs.
3. Enzymes reactions are specific as regards their substrates (reactants) and their products than do
chemical catalysts.
4. Enzymes have capacity for regulation. Catalytic activities of many enzymes vary in response to
their concentrations of substances other than their substrates and products through allosteric
control, covalent modification and variation of the amount of enzymes synthesized.

The study of enzymes is called enzymology. It has come a long way just as the history of Biochemistry.
Both of them evolved together from the 19th Century investigations of fermentation and digestion.
Around 1810, Joseph Gay-Lussac discovered that ethanol and CO2 are the principal products of sugar
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decomposition by yeast. Jacob Berzelius in his theory in 1835 showed that extract of malt known as
diastase (α-amylase) catalyses the hydrolysis of starch more efficiently than does sulphuric acid.

Yet, despite the ability of mineral acids to mimic the effect of diastase, it was the inability to reproduce
most other biochemical reactions in the laboratory that led Louis Pasteur in the middle 19 thC to
propose that the process of fermentation could only occur in living cells. Thus, as was common in his
era, Pasteur assumed that living systems were endowed with a ‘vital force’ that permitted them to
evade the laws of nature governing inanimate matter. Justus von Liebig argued that biological
processes are caused by the action of chemical substances that were then known as “ferments”.

The word ‘enzyme’ is Greek: en = in; zyme= yeast, was coined in 1878 by Fredrich Wilhelm Kuhne in
an effort to emphasize that there is something in yeast , as opposed to the yeast itself, that catalyses
the reactions of fermentation. It was not until 1897 that Eduard Buckner obtained a cell-free yeast
extract that could carry out the synthesis of ethanol from glucose (alcohol fermentation).

In 1894, Emil Fischer discovered that glycolytic enzymes can distinguish between stereoisomeric
sugars which lead to the so-called ‘lock and key hypothesis’: i.e. the specificity of an enzyme (lock) for
its substrate (key) arises from the geometrically complementary shapes. In 1926, James Sumner
crystallized, for the first time, jack bean Urease, which catalyses the hydrolysis of urea to ammonia
and carbon dioxide (NH3) & CO2 and demonstrated that these crystals consist of proteins. This was
later confirmed when John Northrop and Moses Kunitz showed that there was a direct correlation
between the enzymatic activities of crystalline pepsin, trypsin and chymotrypsin and the amounts of
protein present. This proved that generally enzymes are proteins although recently it has been shown
that there are a few RNA molecules with enzymic activity called RIBOZYMES.

In 1963, the first amino acid sequence of an enzyme – bovine pancreatic ribonuclease A was reported
in its entirety. This was followed in 1965 with report of the first X-ray structure of hen egg white
lysosyme. Other enzymes have been purified and characterized since then.

PROPERTIES OF ENZYMES

1. Enzymes have immense catalytic powers.

Most biological reactions cannot take place in the absence of enzymes.

Even the simple reaction: CO2 + H2O H2CO3 is catalyzed by carbonic anhydrase.

2. Enzymes are highly specific.

An enzyme catalyzes a single reaction or a group of related reactions. This high degree of specificity
exhibited by these biological catalysts is one of their remarkable characteristics and seems to explain
their immense catalytic powers.

Enzymes exhibit relative specificity, absolute specificity, and stereo specificity.

(a) Absolute specificity

Glucokinase catalyses only the transfer of phosphoryl group from ATP to glucose to form glucose-6-
phosphate. This is absolute specificity. Absolute specificity in enzymes is the characteristic that makes
enzyme catalyze only one reaction. The enzyme Glucokinase catalyzes only the above reaction and no
other. Urease catalyzes the hydrolysis of urea to carbon dioxide (CO2) and water (H2O) only etc.
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(b) Relative specificity

Relative specificity is also called relative group catalysis. This means the enzyme has the ability to
catalyze group of closely related reactions, e.g. Hexokinase catalyzes the transfer of phosphoryl group
from ATP to hexose sugars like glucose, galactose, mannose, etc. Therefore the enzyme hexokinase
exhibits relative specificity over those sugars. Unlike the Glucokinase which catalyzes the transfer of
phosphoryl group to glucose only, hexokinase catalyzes transfer to glucose, galactose, mannose, etc.

ATP + Glucose Glucose-6-Phosphate + ADP

Hexokinase has relative group specificity, while glucokinase has absolute specificity.

(c) Stereospecificity

This is ability of an enzyme to differentiate between enantiomeric forms of the same type of amino
acid. For instance, α-amino acid and β-amino oxidases are stereospecific enzymes because they are
able to differentiate between enantiomers of the same type of amino acid, e.g. L-amino acid oxidase
catalyzes the oxidation of a variety of amino acids bt not D-configuration. The same applies to D-
amino oxidase which recognizes the D-configuration but not the L-configuration. Another example is
Fumarase which can hydrate fumaric acid (Fumarate) but not maleic acid which are geometric
isomers.

3. Activities of enzymes are regulated.

Living organisms, in order to ensure the preservation of their internal environment (homeostasis), possess
means of regulating the rate of metabolic reactions. Since most, if not all of these metabolic reactions are
catalyzed by enzymes, the regulation of the amount and activity of the enzymes is therefore essential in the
preservation of homeostasis.
Some enzymes are synthesized in the inactive form, to be activated at the appropriate time, e.g., include the
enzymes of blood clotting in which prothrombin is converted to thrombin, fibrinogen to fibrin, etc. Both
thrombin and fibrinogen are in the inactive forms and are converted to their active forms, thrombin and fibrin
during blood clotting.
Other examples include the enzymes of digestion which are synthesized in the inactive forms in the pancreas
and activated by peptide cleavage in the small intestine. The inactive forms are called zymogens or
proenzymes. The following are examples:

Zymogen Active enzyme

Pepsinogen Pepsin
Trypsinogen Trypsin
Chymotrypsinogen A Chymotrypsin A.
Pro-Elastase Elastase

Some enzymes of glycogen synthesis and degradation are regulated by phosphorylation of a specific serine
residue on the enzyme. This modification could be reversed by hydrolysis.

4. Enzymes transform different types of energy

The chemical energy inherent in food could be converted to ATP in the mitochondria. The chemical
bond energy of ATP is utilized in many ways as exemplified in muscle contraction and active
transport. Light energy is converted to chemical energy (photosynthesis). All these processes are
mediated by enzymes.

5. Enzymes are catalysts and they accelerate the forward and backward reaction by definite
factors. Enzymes alter the rates of chemical reactin but does not alter the reaction equilibrium. It
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means that whether an enzyme is present or not, the equilibrium concentration of B is 100 times
greater than A. Enzymes increase the rate at which equilibrium is attained.

6. Enzymes reduce the activation energy of reaction catalyzed by them.

A chemical reaction: A B goes through a transition state that has a higher energy than
either A or B. The rate of reaction is dependent on the concentration of molecules that acquired
enough energy and enter into the transition state.

Enzymes accelerate reactions by reducing the activation energy of the reaction pathway, so thst more
molecules will enter the transition state. They do this by creating a new pathway through which the
reacting molecules could enter into the transition state.

CHEMICAL NATURE OF ENZYMES

Almost all enzymes, except a few RNA molecules with enzymatic activity called ribozymes, are
proteins with large mol.wt. Enzymes with only one polypeptide chain in which the active site resides in
their structure are called MONOMERIC enzymes, eg. Ribonuclease, lysozyme, papain, trypsin,
carboxypeptidase A. They are hydrolytic and usually synthesized as inactive enzymes or zymogens in
the ribosomes before transfer to the GIT where they are rapidly converted to active forms.

Zymogen Active enzyme

Pepsinogen Pepsin
Trypsinogen Trypsin
Chymotrypsinogen A Chymotrypsin A.
Pro-Elastase Elastase

Enzymes are classified as OLIGOMERIC, if they consist of a fascinating combination of polypeptide

chains to form a catalytically active enzyme e.g. Lactate Dehydrogenase (LDH), Hexokinase (Hk).
Each single chain is called a subunit. When many different enzyme-catalyzing sites are engaged in a
sequential series of reactions in the transformation of a substrate to products, it is called a MULTI-
ENZYME COMPLEX. It becomes inactive when fractionated into smaller units, e.g. fatty acid
synthase, carbamoyl phosphate synthetase II, Pyruvate dehydrogenase (PDH), prostaglandin
synthase. Catalytic activity of enzymes depends on the integrity of their protein native protein
conformation. If an enzyme is denatured or dissociated into its subunits, catalytic activity islost. If an
enzyme is broken down into component amino nacids, its catalytic activity is always destroyed. Thus
the primary, secondary, tertiary and quaternary structures of protein enzymes are essential to their
catalytic activity.

Cofactors:

Some enzymes require an additional chemical component called cofactor- either one or more
inorganic ions such as Fe2+, Mg2+, Mn2+, or Zn2+ or a complex organic or metalloorganic molecule
called co-enzyme or one or more metal ions for activity.

Cofactors are small non-protein organic molecules or metal ions that work with enzymes to enhance
catalysis. There are three (3) types of cofactors: Coenzyme, prosthetic group and metal ion. A
complete catalytically active enzyme with its bound co-enzyme and/or metal ions is called a
HOLOENZYME. The protein part of such an enzyme is called an APOENZYME (APOPROTEIN).
The complete structure of an enzyme is as follows:
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Holoenzyme = Apoenzyme +Co-enzyme or Prosthetic group or Metal ion

Prosthetic Group:

This is a non-protein organic substance or metal ion tightly bound to the apoenzyme, e.g. in the
cytochromes, haem (heme) is a prosthetic group tightly bound to globin in haemoglobin.

Co-enzyme:

This is a dialyzable (loosely bound) non-protein organic molecule with which certain enzymes
function. Co-enzymes frequently contain the B-vitamins as part of their structure. Co-enzymes are
active forms of vitamins.

Vitamin Coenzyme (active form) Reaction mediated

Thiamine (B1) Thiamine pyrophosphate (TPP) Carboxylation reactions
Riboflavin (B2) Flavin Adenine Dinucleotide (FAD); Oxidation-Reduction
Flavin Mononucleotide (FMN)
Pyridoxine (B6) Pyridoxal Phosphate (PP) Amino group transfer
Nicotinic acid (Niacin) Nicotinamide Adenine Dinucleotide (NAD) Oxidation-Reductions
Nicotinamdie Adenine Dinucleotide
Phosphate (NADP)
Panthotenic acid Co-enzyme A Acyl group transfer
Biotin Biocytin Carboxylations reactions
Folic acid Tetrahydrofolate (TH4) One-Carbon transfer
Vit. B12 Deoxyadenosyl cobalamin Intra-molecular rearrangement
Vit C (Ascorbic acid) Unknown Hydroxylation reactions
Vit. A Retinal Vision, Growth, Reproduction
Vit D. 1,25-dihydrocholecalciferol Ca2+& P metabolism
Vit. E Unknown Lipid anti-oxidant
Vit K Unknown Blood clotting

Sometimes a coenzyme could behave as a second substrate, e.g. phosphorylation of a sugar by ATP.

Metal ion:
Many enzymes require presence of a metal ion for activity. The metal may serve a structural role or
function or as a Lewis acid (a positive ion that can bind to unpaired electrons) or as an electron
acceptor/donor in oxidation-reduction reactions. It is described as substrate-bridged (Enz-S-M) if the
metal interacts first with the substrate before interacting with the enzyme. For instance, in ATP-
dependent reactions, ATP4- interacts with Mg2+ to form Mg-ATP2- complex which then interacts with
the enzyme to form an Enz-S-M ternary complex as can be seen in reactions involving kinases. In
another instance, metals that chelate the substrate or ATP to the enzyme to form metal-bridge
ternary complexes (Enz-M-S). There are enzymes that contain tightly bound transition metals such
as Zn2+ or Fe2+ that facilitate binding of substrate and promoting electrophilic (nucleophilic catalysis)
at the site of bond cleavage or stabilizing intermediates in the reaction pathway called
metalloenzymes. For instance, alcohol dehydrogenase has an enzyme-bound Zn 2+ just like
carboxypeptidase and thermolysin. Metals also involve in redox reactions as acceptor of electrons
such as copper and iron (transition metals) because of their incomplete outer shell orbital electrons.

OTHER DEFINITIONS

Rate of a reaction: This is the change in the amount (moles, grams) of starting materials or products
per unit time.
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Substrate: It is the molecule on which the enzyme acts upon. In reversible reactions, the products of
the forward reaction are the substrates of the backward reaction. A +B C+D

Substrate binding site: This is a particular arrangement of chemical groups residing in a particular
region on the enzyme surface that is specifically formulated to bind a specific substrate.

Active Site: This is a site that possesses the machinery, in the form of particular chemical groups that
is involved in catalyzing the reaction under consideration. It may be within the Substrate binding site
or outside it but may be contiguous to it in the primary sequence. The chemical groups involved in
both binding of substrates and catalysis are often part of the side chains of the amino acids of the
apoenzyme.

Isoenzymes; These are electrophoretically distinguishable variants of the enzyme but they all catalyze
the same reaction. For example, the isoenzymes of Lactate Dehydrogenase: It is a tetrameric protein
enzyme that has five (5) variants located in the liver as L 4 and in the muscle as M4 with the variants
L3H1, L2H2 and L1H3 found in other tissues. They all catalyze the reduction of pyruvate to Lactate:

Pyruvate + NADH + H+ Lactate + NAD+

How enzymes work: Energy Levels

For most chemical reactions to proceed, some form of energy is needed. It is called "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. See figure below.

Enzymes 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.

Factors Affecting Enzyme Activity

1. Enzyme Concentration
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In the presence of excess amount of substrate, increase in enzyme concentration increases the rate of
reaction a shown in fig. 4 above: The amount of enzyme present in a reaction is measured by the
activity it catalyzes. The relationship between activity and concentration is affected by many factors
such as temperature, pH, etc. An enzyme assay must be designed so that the observed activity is
proportional to the amount of enzyme present in order that the enzyme concentration is the only
limiting factor. It is satisfied only when the reaction is zero order. The same shape of graph will be
obtained when the concentration of the product of an enzymatic reaction is plotted against time
(Figure 6.)

2. Substrate Concentration

It has been shown experimentally that if the amount of the enzyme is kept constant and the substrate
concentration is then gradually increased, the reaction velocity will increase until it reaches a
maximum. After this point, increases in substrate concentration will not increase the velocity (delta
A/delta T). This is represented graphically in Figure 8.

When maximum velocity had been reached, all of the available enzyme has been converted to ES, the
enzyme substrate complex. The velocity at this point on the graph is designated Vmax. Using this
maximum velocity and equation (7), Michaelis developed a set of mathematical expressions to
calculate enzyme activity in terms of reaction speed from measurable laboratory data.

The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity.
This is shown in Figure 8. Using this constant and the fact that Km can also be defined as:

Km=K-1 + K2 / K+1

K+1, K-1 and K+2 being the rate constants from equation (7). Michaelis developed the following
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The size of Km tells us several things about a particular enzyme.

 A small Km indicates that the enzyme requires only a small amount of substrate to become
saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations.
 A large Km indicates the need for high substrate concentrations to achieve maximum reaction
velocity.
 The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently
assumed to be enzyme's natural substrate, though this is not true for all enzymes.

3. Effects of Inhibitors on Enzyme Activity

Enzyme inhibitors are substances which alter the catalytic action of the enzyme and consequently slow
down, or in some cases, stop catalysis. There are three common types of enzyme inhibition -
competitive, non-competitive and substrate inhibition.

Most theories concerning inhibition mechanisms are based on the existence of the enzyme-substrate
complex ES. As mentioned earlier, the existence of temporary ES structures has been verified in the
laboratory.

Competitive inhibition occurs when the substrate and a substance resembling the substrate are both
added to the enzyme. A theory called the "lock-key theory" of enzyme catalysts can be used to explain
why inhibition occurs.

The lock and key theory utilizes the concept of an "active site." The concept holds that one particular
portion of the enzyme surface has a strong affinity for the substrate. The substrate is held in such a
way that its conversion to the reaction products is more favorable. If we consider the enzyme as the
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lock and the substrate the key (Figure 9) - the key is inserted in the lock, is turned, and the door is
opened and the reaction proceeds. However, when an inhibitor which resembles the substrate is
present, it will compete with the substrate for the position in the enzyme lock. When the inhibitor
wins, it gains the lock position but is unable to open the lock. Hence, the observed reaction is slowed
down because some of the available enzyme sites are occupied by the inhibitor. If a dissimilar
substance which does not fit the site is present, the enzyme rejects it, accepts the substrate, and the
reaction proceeds normally.

Non-competitive inhibitors are considered to be substances which when added to the enzyme alter the
enzyme in a way that it cannot accept the substrate. Figure 10.

Substrate inhibition will sometimes occur when excessive amounts of substrate are present. Figure 11
shows the reaction velocity decreasing after the maximum velocity has been reached.

Additional amounts of substrate added to the reaction mixture after this point actually decreases the
reaction rate. This is thought to be due to the fact that there are so many substrate molecules
competing for the active sites on the enzyme surfaces that they block the sites and prevent any other
substrate molecules from occupying them. This causes the reaction rate to drop since all of the enzyme
present is not being used.

4. Temperature Effects

Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is
raised. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to
100%.
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Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in

the results. Because enzymes are proteins, they are adversely affected by high temperatures. As shown
in figure below, the reaction rate increases with temperature to a maximum level, then abruptly
declines with further increase of temperature. Because most animal enzymes rapidly become
denatured at temperatures above 40°C, most enzyme determinations are carried out somewhat below
that temperature.

5. Effects of pH

Enzymes, like proteins made up of amino acids, are affected by changes in pH. The most favorable pH
value - the point where the enzyme is most active - is known as the optimum pH as shown below:

Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is
also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH
optimal stability. The optimum pH value will vary greatly from one enzyme to another, as shown
below:

pH for Optimum Activity

Enzyme pH Optimum
Lipase (pancreas) 8.0
Lipase (stomach) 4.0 - 5.0
Lipase (castor oil) 4.7
Pepsin 1.5 - 1.6
Trypsin 7.8 - 8.7
Urease 7.0
Invertase 4.5
Maltase 6.1 - 6.8
Amylase (pancreas) 6.7 - 7.0
Amylase (malt) 4.6 - 5.2
Catalase 7.0

In addition to temperature and pH there are other factors, such as ionic strength, which can affect the
enzymatic reaction. Each of these physical and chemical parameters must be considered and optimized
in order for an enzymatic reaction to be accurate and reproducible.

Kinetics Two-substrate reactions

There are two broad mechanisms of enzyme interaction with two substrates: Sequential and Ping pong.

Sequential mechanism: In sequential mechanism, both substrates bind to the enzyme in a sequential manner
to form a ternary complex. With substrates A, and B, the ternary complex would be E-A-B (A,B, C, .. are
substrates and P, Q, R….. are products). If substrate A binds prior to B, then it is called Ordered –sequential
reaction, E.g., oxidation of alcohol by NAD + to produce Ethanal (acetaldehyde) and NADH + H + while if either
A or B can bind first, the reaction is called random-sequential reaction (e.g. phosphorolytic cleavage of
glucose unit from glycogen by phosphorylase using inorganic phosphate to produce glucose-1-phosphate.

Ping-Pong mechanism

In this mechanism, substrate A binds to the enzyme and is converted to P, leaving a modified enzyme which
binds substrate B that is converted to product Q. Enzymes that use Ping-Pong kinetics are modified by the
substrate, e.g. Kinases that phosphorylate the enzyme, ADP dissociates and the second substrate binds to the
phosphorylated enzyme followed by phosphate transfer to produce the phosphorylated acceptor. Enzymes
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that use covalent catalysis or have coenzymes that do not dissociate employ ping-pong reactions. An example
is carboxylation of acetyl-CoA to malonyl-CoA by malonyl-CoA synthetase in the presence of ATP.

Sequential Ping-Pong

E + A EA E + A EA E + B EB
EA + B EAB EA E’P EB E’P
EAB EPQ E’P E’ + P E’P E’ + P
EPQ EP + Q E’ + B E’B E’ + A E’A
EP E + P E’B E +Q E’A E +Q

Mechanism of action of enzymes

The making and breaking of a chemical bond by an enzyme is preceded by the formation of an
enzyme-substrate complex (ES complex). The existence of sn enzyme-substrate complex is inferred
from:

(a) The high degree of specificity exhibited by enzymes

(b) The shape of the velocity (v) versus substrate concentration ([S] curve
(c) The fact that the substrate frequently protect enzymes from inactivation
(d) Stable enzyme-substrate complexes have been isolated.

E + S ES EP E + P
Enzyme Substrate Enzyme-substrate Enzyme-Product Enzyme Product
complex complex

In 1894, Emil Fischer put up the Lock and Key analogy for enzyme catalyzed reactions. He assumed
that the Active Site, which is complimentary