BIOCHEMISTRY

Enzyme Chemistry Part 1

Dr. Billones
BIOMEDICAL IMPORTANCE OF ENZYMES  Organic cofactors are usually vitamins. Majority are coming
 Life is not possible without enzymes from the water soluble B vitamins
 Some enzyme deficiency may lead to diseases
 Enzymes are therapeutically targeted by some drugs
 May be used as diagnostic markers
 Utilized as analytical reagents in research

WHAT ARE ENZYMES?

1) Enzymes are proteins which accelerate chemical reactions
BUT not all enzymes are proteins, some are RNA called ribozymes

2) Enzymes have an active site on its structure. Active site is the region
in the enzyme structure where a substrate binds
Characteristics of Active Site:
1. It constitutes only a small part of the total volume of
the enzyme
2. It is a three-dimensional entity
3. It binds the substrate through relatively weak forces
or bonds
4. It is a cleft or a crevice in the enzyme molecule
Organic cofactors can be further divided into two:
1. Prosthetic groups- organic cofactor that is bound
tightly to the enzyme. Hard to be removed from the
enzyme
2. Coenzyme- organic cofactor that is loosely bound to
the protein enzyme. Can easily be detached from the
enzyme

Parts of the Active Site Coenzyme Importance

a) Binding Site  Assist in the cleavage of the substrate by acting as an
Part where the substrate adheres or attaches to the acceptor for one of the cleavage products or to assist in
enzyme the transformation of the substrate

b) Catalytic Site PRINCIPAL COENZYMES

Composed of amino acids that bring about the Coenzyme Vitamin Source Function
formation or breakage of bond or causes the NAD, NADP Niacin Electron transfer
transformation of the substrate FMN, FAD Riboflavin Electron transfer
Pantothenic Acyl group
Coenzyme A
acid transfer
TPP Thiamin Aldehyde transfer
Pyridoxal Amino group
Pyridoxine
phosphate transfer
Carbon dioxide
Biotin Biotin
transfer
Tetrahydrofolic One carbon
Folic acid
acid (FH4) transfer

3) Enzymes exhibit a high degree of specificity for their substrate

APOENZYME vs. HOLOENZYME
4) For enzymes to be activated they need to a Cofactor which can be
 Apoenzyme- is the inactive form of an enzyme. The protein portion
organic or inorganic
of the enzyme system
 Inorganic cofactors are usually minerals
Why is it inactive? It is inactive because there is no cofactor
attached to it. So when a cofactor attaches to the apoenzyme, it
becomes catalytically active

 Holoenzyme- it is the active form of enzyme with its non-protein

component
Apoenzyme (protein) + Cofactor (non-protein)

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 BIOCHEMISTRY
Enzyme Chemistry Part 1
Dr. Billones
GENERAL NATURE OF ENZYMES ENZYME NOMENCLATURE
1. All enzymes are proteins  A conventional way of naming enzymes is by adding the suffix “ase”
o Note: Some types of RNA can act like enzymes, usually catalyzing the to the substrate or to the type of reaction they catalyze
cleavage and synthesis of phosphodiester bonds. RNAs with catalytic
activity are called ribozymes Substrate + “ase”

2. Enzymes are not consumed, destroyed or produced during the process

of catalysis. They are regenerated at the end of the chemical reactions

3. Enzymes do not cause reactions to take place. They enhance the rate
of the reactions, without enzyme, reactions tend to be slower

4. Enzyme catalysis involves the formation of enzyme-substrate (ES)

complex

Urea in the presence of water would yield ammonia. The

substrate urea and the enzyme would be urease

o At a constant concentration of an enzyme, the reaction rate increases

with increasing substrate concentration until a maximal velocity is
reached. In contrast, uncatalyzed reaction do not show this
saturation point

5. The substrate is bound to a specific region of the enzyme called active

site. Most enzymes are highly selective in their binding of substrates The name of the enzyme is pyruvate decarboxylase. The pyruvate
here has been decarboxylated. The carboxyl group has been
cleaved off and liberated in the form of CO2
6. Enzymes lower the activation energy required for a chemical reaction
to take place
Reaction + “ase”
o Activation energy: is the amount of energy that must be supplied
before there is sufficient interaction between reactants to form a
product

EXCEPTIONS to enzyme nomenclature:

 There are proteolytic enzymes that does not end with the suffix
“ase” but rather with suffix “in”
 Examples: Trypsin, Chymotrypsin, & Pepsin
 Restriction endonuclease. These are enzymes that cleave DNA in a
very specific manner. They are named after the bacterial sources they
come from
 Examples: EcOR1 and Mst2
o Picture on the left, in color red, in the absence of enzyme, the energy
needed to be supplied in order to create a product is higher. So the
activation energy here is higher
o Picture on the right, in color green, in the presence of enzyme, the
energy needed to create a product is lower. So what enzyme does is
to lower the activation energy

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Enzyme Chemistry Part 1
Dr. Billones
SIX MAJOR CLASSES OF ENZYMES 5. Isomerases
1. Oxidoreductase  Catalyze intramolecular rearrangement
 Catalyze oxidation-reduction reactions. Functions in the a) Racemases- interconvert L and D stereoisomers
transfer of electrons b) Mutases- produce aldehydes via elimination reactions
a) Oxidases- use oxygen as an electron acceptor
b) Dehydrogenases- use molecules other than oxygen (e.g.
NAD,FAD) as an electron acceptor
c) Oxygenases- directly incorporate oxygen into the substrate
d) Peroxidases- use H2O2 as an electron acceptor

6. Ligases
 Catalyze reaction in which two molecules are joined
a) Carboxylases- use CO2 as a substrate
b) Synthetases- link two molecules via an ATP-dependent
2. Transferases reaction
 Catalyze the transfer of a functional group from one molecule
to another
a) Methyltransferases- transfer one-carbon units between
substrates
b) Aminotransferases- transfer NH2 from amino acids to keto
acids
c) Kinases- PO-3 from ATP to a substrate
d) Phosphorylases- transfer PO-3 from inorganic phosphate to
a substrate Major Classes of Enzymes with Examples of Each

3. Hydrolases
 Catalyze bond cleavage by the introduction of water
a) Phosphatases- remove PO-3 from a substrate
b) Phosphodiesterases- cleave phosphodiester bonds in
nucleic acids
c) Proteases- cleave amide bonds (peptide bonds) in proteins
d) Esterases- cleave ester bonds such as those in
carbohydrates
e) Glycosidases- cleaves glycosidic bonds such as those in
carbohydrates

MECHANISM OF ENZYME ACTION

 Enzyme bind substrate at the active site/catalytic site
 Active site fits shape of substrate
4. Lyases
 It forms a enzyme-substrate (ES) complex
 Add (or remove) the elements of water, ammonia or
 Association between enzyme and substrate is temporary so after
carbon dioxide to (or from) double bonds
forming products they can regenerate the free enzyme
a) Decarboxylases- produces CO2 via elimination reactions
b) Aldolases- produce aldehydes via elimination reactions
c) Synthases- link two molecules without involvement of
 Aside from the active site, there also what we call an allosteric site
ATP
 Allosteric site is any site other than the active site.

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Enzyme Chemistry Part 1
Dr. Billones
TYPES OF ENZYME SPECIFICITY 2. Conversion of an enzyme precursor
a) Relative specificity- enzymes act on a particular kind of covalent Specific proteolysis is a common method of activating
bond in closely related substrate enzymes and other proteins in biological system
b) Absolute specificity- enzymes act only on one substrate or chemical Example:
reaction The generation of trypsin from trypsinogen leads to
c) Optical specificity- enzyme act only on one type of isomer the activation of other zymogens

The specificity of an enzyme is determined by the functional groups of Intracellular and Extracellular Enzymes
the substrate, the functional groups of the enzyme, and the physical  Intracellular enzymes are synthesized and retained in the cell for
proximity of these functional group the use of cell itself. They are found in the cytoplasm, nucleus,
mitochondria and chloroplast
Two theories have been proposed to explain the specificity of enzyme action: Example: Oxireductase catalyzes biological oxidation. Enzymes
involved in reduction in the mitochondria
1. Lock and Key Theory (Fischer Template Hypothesis)
 Proposed by Emil Fischer in 1894  Extracellular enzymes are synthesized in the cell but secreted from
 Lock and key hypothesis the cell to work externally
assumes that the active site of an Example: Digestive enzyme produced by the pancreas, are not
enzyme is rigid in its shape used by the cells in the pancreas but are transported to the
 There is no change in the active duodenum
site before and after a chemical
reaction FACTORS AFFECTING ENZYME ACTIVITY AND REACTION VELOCITY
 The enzyme’s active site is 1. Effect of Temperature
complementary in conformation  All chemical reaction get accelerated with rise in
to the substrate, so that the temperature whether mediated by enzymes or not
enzyme and substrate recognize  There is an optimal temperature at which the reaction is
one another most rapid
 This explains how enzyme specificity happens for a particular  An optimum temperature is usually reached at 40oC to 50oC
substrate for animal enzymes, whereas plant enzymes it is higher,
usually at 50oC to 60oC
2. Induced-Fit Theory (Koshland Model)
 Above this, the rate decreases because the enzyme is
 Proposed by Daniel Koshland in 1958 denatured
 According to this theory, exposure of an enzyme to substrate causes  Temperature also give a bell-shaped curve
a change in enzyme, which causes the active site to change its shape
which allow enzyme and substrate to bind
 The enzyme changes shape after binding with the substrate, so that
the conformation of substrate and enzyme is complementary only
after binding
 Explains enzymes broad specificity

ENZYME ACTIVATION
 Enzyme activation is defined as the conversion of an inactive form of
an enzyme to active form which processes the metabolic activity
 There are 2 types of enzyme activation
1. Activation by cofactors
Many enzymes are activated by cofactors.
Examples:
DNA polymerase is a holoenzyme that catalyzes the
polymerization of deoxyribonucleotide into a DNA
strand. It uses Mg- ion for catalytic activity.
Horse liver dehydrogenase uses Zn- ion for its
activation
Picture above:
Most of the human enzymes, the optimum temperature is 36.5OC to 37.5oC.
So this means that at this temperature, the activity of the enzyme is
maximum. If we lower the temperature we inactivate the enzyme. If we
increase the temperature, we denature the enzyme.
2. Effect of pH
 Enzyme reactions are influenced by carrying hydrogen ion
concentration
 The optimum pH is that pH at which a certain enzyme causes
a reaction to progress most rapidly
 On either side of the optimum, the reaction rate is lower
since at certain pH’s enzyme maybe inactivated or
denatured

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Enzyme Chemistry Part 1
Dr. Billones
 The pH of the medium also affects the ionization of ENZYME REGULATION
functional groups of the enzyme or the substrate 1. Irreversible Covalent Modification/Zymogen Cleavage
 Although ideally the pH activity curve is bell-shaped, not all 2. Allosteric Regulation
enzymes exhibit the ideal 3. Reversible Covalent Modification
4. Genetic Control

1.) Irreversible Covalent Modification/Zymogen Cleavage

 Proteolytic enzymes are stored as zymogens or proenzymes
 Zymogen = inactive
 Zymogen activation needs to undergo cleavage process
 Zymogens are inactive forms of enzymes that become active only
after being cleaved at specific site in their polypeptide chain by
specific proteases

Examples of Zymogens:

Picture Above:
The optimum pH for most human enzymes is around 7-7.45 pH. Enzymes
denature if they are outside their pH range. In the example above, pepsin’s
activity is optimum at an acidic pH while human amylase activity is optimum
at neutral pH and lastly, trypsin work’s best at an alkaline pH.

3. Effect of enzyme concentration

 The velocity of an enzyme reaction is directly proportional to
the concentration of the enzyme, provided that the
substrate is present in excess
 This is particularly true at the beginning of the reaction, but
it may not hold true as the reaction continues
4. Effect of substrate concentration
 The rate or velocity of reaction (v) is the number of substrate
molecule converted to product per unit of time and is usually
expressed as µmoles produce formed per minute
 The rate of an enzyme-catalyzed reaction increases with
substrate concentration until a maximal velocity (Vmax) is
reached
 The levelling off of the reaction rate at high substrate
concentrations reflects the saturation with substrate of all
available binding sites on the enzyme
2.) Allosteric Regulation
 These enzymes are regulated by molecules called effectors (also
called modulators) that bind non-covalently at a site (allosteric site)
other than the active site
 The presence of an allosteric effect can alter the affinity of the
enzyme for its substrate or modify the maximum catalytic activity of
the enzyme or both
 Negative effectors inhibit enzyme activity, whereas those that
increase enzyme activity are called positive effectors
 When the substrate itself serves as an effector, the effect is said to
be homotropic
 Most often an allosteric substrate function as a positive effector
 The effector maybe different from the substrate, in which case the
effect is said to be heterotropic
 Allosteric enzymes are usually found in the early steps (1st or 2nd
reaction) in a metabolic pathway
 They are also called committed enzymes or rate-limiting enzymes
 They are subject to inhibition by the end-product of the metabolic
pathway (negative feedback inhibition)
 This prevents unnecessary production of an excess end-product by
shutting down the pathway until more is needed

Picture on the left:

As we increase the substrate
concentration, the enzyme
activity (reaction rate) also
increases. But there is a point
Picture above: How Allosteric Regulation Happens
called the saturation point,
The enzyme has an active site and allosteric site. The substrate combines
where it has already reached with the active site and it creates a chemical reaction (turning green) which
the maximum substrate then form products
concentration. This means that Continuation of Allosteric Regulation on next page…..
all enzymes are bound to a
substrate

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Enzyme Chemistry Part 1
Dr. Billones
Picture Above: Part 2
When there is an abundance of products, some of its products will bind to
the allosteric site which causes a change in the active site (encircled in red)
Picture Above: Part 3
The binding of product to the allosteric site causes the enzyme to be
inhibited, therefore shutting down the pathway. So what happens is a
feedback inhibition. This means that when the final product accumulates in
abundance, the excess final product will bind to the allosteric site, specifically
affects the first enzyme in the series of reaction making it the rate-limiting
enzyme. So when an enzyme is inhibited, less products will be formed
Characteristics of Allosteric Enzymes:
a) They have a very high molecular weight
b) They are complex oligomeric proteins
c) They are more difficult to purify compared to ordinary enzymes
d) They do not conform to the classical Michaelis-Menten
relationship
3.) Reversible Covalent Modification
 In this class of regulatory enzymes, the active and inactive forms are
interconverted by covalent modifications of their structures that are
catalyzed by other enzymes
 In certain enzymes, the addition of a phosphate group
(phosphorylation) to a specific amino acid residue (usually serine,
tyrosine, threonine) by specific protein kinases dramatically
enhances or depresses activity
 The phosphorylated enzyme may be dephosphorylated by specific
phosphatases
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Picture above: Glycogen phosphorylase
An example of covalent modification can be found in glycogen
phosphorylase. In the period of starvation, there is a decrease in the glucose
level in the blood so there will be a release of the hormone glucagon.
Glucagon will activate kinases. Kinases transfer phosphate group from ATP
to activate glycogen phosphorylase, so it becomes phosphorylated. Since
glycogen phosphorylase is activated, it can now catalyze glycogenolysis
(breaking down of glycogen to increase glucose level).
If there is already enough level of glucose in the blood, there will be a release
of the hormone insulin. Insulin will activate phosphatase, which functions to
remove the phosphate group from the glycogen phosphorylase. If phosphate
group is removed (dephosphorylation), it will inactivate the enzyme
glycogen phosphorylase. So from glycogenolysis, the body goes into
glycogenesis (creation of glycogen storage)
 Reversible covalent modification of enzyme activity via
phosphorylation/dephosphorylation
 Phosphorylation catalyzed by protein kinases
 Dephosphorylation catalyzed by protein phosphatases
4.) Genetic Control
 Cells can also regulate the amount of enzyme present by altering the
rate of enzyme synthesis
 The increase (induction) or decrease (repression) synthesis of the
enzyme leads to an alteration in the total population of active sites,
rather than influencing the efficiency of existing enzyme molecules
 The mechanism seems to be stimulation or inhibition of
transcription of the gene in the nucleus that codes for the protein
enzyme
 Exemplified by Lac Operon
Continuation of Genetic Control next page…..
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Enzyme Chemistry Part 1
Dr. Billones

Picture Above:
 Lac Operon encodes for enzyme lactase
 Enzyme lactase is produced ONLY if substrate lactose is present
 Lac operon is turned OFF without substrate lactose
 Without lactose, repressor binds to operator
 Therefore, RNA polymerase is unable to bind to promoter
 Lac operon is NOT transcribed into mRNA and NOT translated in
lactase
 When lactose binds to the active repressor protein, it will be
inactivated
 RNA polymerase will now be able to bind to the promoter region
 Lac operon will now be transcribed into mRNA and mRNA will be
translated into lactase

REFERENCES

 Biochemistry Manual (2018)

 Dr. Bravo PPT on Enzyme (For Pictures)
 Dr. Billones Recording

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