The nature, properties and classification of enzymes and

cofactors.

Enzymes are biological molecules that act as catalysts in chemical

reactions within living organisms. These macromolecules significantly
accelerate the rate of reactions, ensuring the efficient functioning of all
cellular processes.

Enzymes are primarily composed of proteins, with some rare exceptions

being ribozymes, which are catalytic RNA molecules. The protein
structure of an enzyme folds into a unique three-dimensional shape,
creating a specialized region called the active site. This active site serves
as the binding pocket for specific molecules, called substrates, that the
enzyme acts upon.

Characteristics of Enzymes

● Protein-based: Enzymes are largely comprised of protein subunits

linked by peptide bonds. The specific sequence of amino acids

determines the enzyme's unique structure and function.

● Highly Specific: Each enzyme has a specific shape and charge

distribution that allows only certain substrates to bind to its active

 site, ensuring reaction selectivity. This specificity is often likened to
a "lock-and-key" model.

● Catalytic Powerhouse: Enzymes significantly accelerate reaction

rates by lowering the activation energy required to initiate the

reaction. This allows reactions to occur under physiological
conditions (temperature and pH) within cells.

● Temperature Sensitivity: Enzymes function at an optimal

temperature range for maximum activity. Deviations from this

range can cause the enzyme structure to denature (unfold), leading
to decreased activity or complete inactivation.

● pH Dependence: Enzymes function most effectively at a specific pH

range (optimum pH). Extreme acidic or basic environments can

alter the enzyme's charge distribution, affecting substrate binding
and catalysis.

● Susceptible to Inhibitors: Certain molecules can bind to enzymes

and inhibit their activity. Competitive inhibitors mimic the substrate

and compete for the active site, while non-competitive inhibitors
bind to a different site on the enzyme, altering its conformation.

● Affected by Cofactors: Some enzymes require additional non-

protein molecules called cofactors for optimal activity. Cofactors

can be inorganic metal ions (e.g., magnesium, zinc) or organic
 molecules (coenzymes). Coenzymes often participate directly in the
catalytic cycle by accepting or donating functional groups during the
reaction.

Classification of Enzymes
Enzymes are classified based on the type of chemical reaction they
catalyze. The six major enzyme classes are:
1. Oxidoreductases: catalyzes the transfer electrons between
molecules. (e.g., dehydrogenase)
AH2+B A+BH2

2. Transferases: catalyzes the transfer of functional groups between

molecules. (e.g., kinase)
A–X+B↔B–X+A

3. Hydrolases: Break down molecules by hydrolysis (using water). (e.g.,

lipase)
A–X+H2O X–OH+A–H

4. Lyases: Cleave bonds between atoms without catalysis (e.g., aldolase)

A–X+B–Y A=B+X–Y
5. Isomerases: Rearrange the atoms of a molecule to create an isomer.
(e.g., epimerase, phosphoglucomutase)
ACis A′Trans

6. Ligases: Form new covalent bonds between molecules, often at the

expense of ATP hydrolysis. (e.g., DNA polymerase)
A+B AB

Cofactors
Cofactors are non-proteinous substances that act as helpers of proteins.
Usually, cofactors partner with enzymes to provide chemical
functionalities or binding sites that the enzymes may lack. Without
cofactors, major enzymatic reactions may be halted.

Types of Cofactors
1. Inorganic Cofactors: These are simple metal ions like magnesium
(Mg2+), iron (Fe2+), or zinc (Zn2+). They often participate in
catalysis by stabilizing reaction intermediates or facilitating electron
transfer.
2. Carrier Coenzymes: These are more complex organic molecules,
often derived from vitamins or other essential nutrients.
Coenzymes undergo reversible chemical modifications during the
 reaction cycle, carrying functional groups like hydrogens or
phosphates to facilitate the reaction.
3. Prosthetic groups: These are cofactors tightly bound to an enzyme
at all times e.g. FAD (flavin adenine dinucleotide).

Functions of Cofactors
1. Enhance Enzyme Activity: Cofactors often lower the activation
energy of a reaction, significantly increasing the reaction rate
catalyzed by the enzyme.
2. Specificity: Like enzymes, some cofactors exhibit a degree of
specificity. Certain coenzymes might work with a particular group
of enzymes involved in similar reactions.
3. Regeneration: Coenzymes are often recycled after they donate or
accept a functional group. This allows them to participate in
multiple catalytic cycles with their partner enzyme.
4. Dietary Dependence: Many coenzymes are derived from vitamins or
essential nutrients. Deficiencies in these nutrients can lead to
decreased activity of enzymes that rely on those specific
coenzymes.

Mechanism of Enzyme Reaction

Enzymes possess an active site. The active site is a part of the
molecule that has a definite shape and the functional group for the
binding of reactant molecules. The molecule that binds to the enzyme is
referred to as the substrate group. The substrate and the enzyme form an
intermediate reaction with low activation energy without any catalysts.
The basic mechanism of enzyme action is to catalyze the chemical
reactions. The active site of an enzyme draws substrates and catalyzes
the chemical process that produces products, this process forms an
enzyme-substrate complex. The enzyme-substrate complex is the
combination of an enzyme and its substrates.
The reaction requires the collision of any two molecules, as well as the
correct orientation and a sufficient quantity of energy. This energy must
be transferred between these molecules in order to overcome the
reaction’s Activation Energy barrier. Without any catalysts, the substrate
and enzyme produce an intermediate reaction with low activation energy.

pH and Temperature effects on enzymes

Enzymes function at an optimum temperature and pH. The temperature
or pH at which a compound shows its maximum activity is called
optimum temperature. A temperature or pH more than optimum may
alter the molecular structure of the enzymes. Most enzymes have a
characteristic optimum pH at which the velocity of the catalyzed reaction
is maximal.
The pH profile is dependent on a number of factors. As the pH changes,
the ionization of groups both at the enzyme’s active site and on the
substrate can alter, influencing the rate of binding of the substrate to the
active site. These effects are often reversible. For example, if we take an
enzyme with an optimal pH (pHopt) of 7.0 and place it in an environment
at pH 6.0 or 8.0, the charge properties of the enzyme and the substrate
may be suboptimal, such that binding and hence the reaction rate are
lowered. If we then readjust the pH to 7.0, the optimal charge properties
and hence the maximal activity of the enzyme are often restored.

However, if we place the enzyme in a more extreme acidic or

alkaline environment (e.g. at pH 1 or 14), although these conditions may
not actually lead to changes in the very stable covalent structure of the
protein’s primary structure (i.e. its configuration), they may well produce
changes in the conformation (shape) of the protein such that, when it is
returned to pH 7.0, the original conformation and hence the enzyme’s
full catalytic activity are not restored. It should be noted that the
optimum pH of an enzyme may not be identical to that of its normal
intracellular surroundings. This indicates that the local pH can exert a
controlling influence on enzyme activity.

The effects of temperature on enzyme activity are quite complex, and

can be regarded as two forces acting simultaneously but in opposite
directions. As the temperature is raised, the rate of molecular movement
and hence the rate of reaction increases, but at the same time there is a
progressive inactivation caused by denaturation of the enzyme protein.

Thermal denaturation is time dependent, and for an enzyme the

term ‘optimum temperature’ has little real meaning unless the duration of
exposure to that temperature is recorded. The thermal stability of an
enzyme can be determined by first exposing the protein to a range of
temperatures for a fixed period of time, and subsequently measuring its
activity at one favorable temperature (e.g. 25°C).

The temperature at which denaturation becomes important varies from

one enzyme to another. Normally it is negligible below 30°C, and starts
to become appreciable above 40°C. Typically, enzymes derived from
microbial sources show much higher thermal stability than do those from
mammalian sources, and enzymes derived from extremely thermophilic
microorganisms, such as thermolysin (a protease from Bacillus
thermoproteolyticus) and Taq polymerase (a DNA polymerase
from Thermus aquaticus), might be completely thermostable at 70°C and
still retain substantial levels of activity even at 100°C.

Factors Affecting Enzyme Catalysis

1. Concentration of Substrate: In the presence of an enzyme, the rate

of a chemical reaction increases as the substrate concentration rises
until a limiting rate is achieved, after which additional increases in
the substrate concentration have no effect on the reaction. The
enzyme molecules are saturated with the substrate at this point.
The extra substrate molecules are unable to react until the substrate
that has already been bound to the enzymes has reacted and been
released.

2. Concentration of Enzyme: When the enzyme concentration is much

lower than the substrate concentration, the rate of an enzyme-
catalyzed reaction is proportional to the enzyme concentration. This
is true for any catalyst; when the catalyst concentration rises, the
reaction rate rises as well.

3. Temperature: For most chemical reactions, a temperature increase

of 10°C about doubles the reaction rate, according to a well-known
rule of thumb. Even a slight increase in temperature, after a certain
 threshold, induces denaturation of the protein structure and
disruption of the active site, resulting in a decline in reaction rate.

4. Hydrogen Ion Concentration (pH): Most enzymes are proteins, and

they are sensitive to variations in pH or hydrogen ion concentration.
The degree of ionisation of an enzyme’s acidic and basic side
groups, as well as the substrate components, is affected by changes
in pH. The catalytic activity of an enzyme is altered when one of
these charges is neutralised. Over a narrow pH range, an enzyme’s
activity is at its peak. The enzyme’s optimal pH is determined by
the median value of this pH range.

5. Inhibition of Enzymes: Enzymes must occasionally be slowed to aid

and ensure that our bodies’ systems operate appropriately and
efficiently. For example, if an enzyme produces too much of a
product, it must be possible to reduce or stop production. Inhibitors
are required in such situations.
Chemical kinetics and applied thermodynamics
Enzyme kinetics is a branch of biochemistry that deals with the
study of enzymatic reactions and their rates. Understanding enzyme
kinetics is crucial for various fields such as medicine, pharmacology, and
biotechnology.

Key concepts and terms related to enzyme kinetics

1. Enzyme-substrate complex (ES): This is formed when an enzyme
binds to its substrate(s), forming a temporary intermediate
complex. The enzyme-substrate complex is where the actual
chemical reaction takes place.
The mechanism of action of enzyme inhibitors includes a step of enzyme-
inhibitor complex formation (EI complex) that has no (or low) enzyme
activity.

2. Michaelis-Menten kinetics: is a classical model used to describe the

rate of enzymatic reactions. Enzyme reactions do not show simple zero,
first or second order relationships of chemical reactions. Instead, the
reaction reaches a limiting or saturation rate. This behaviour is governed
by the Michaelis-Menten equation and the two characteristic constants
associated with this equation are Vmax and Km.
3. Vmax: The maximum velocity or rate of an enzymatic reaction, which
occurs when all enzyme active sites are saturated with substrate.
4. Km: (Michaelis constant): is a measure of the affinity of an enzyme for
its substrate. A lower Km value indicates a higher affinity, meaning the
enzyme can achieve half of its maximum velocity at lower substrate
concentrations.
The Michaelis-Menten equation relates the initial reaction velocity (V) to
the substrate concentration ([S]), the maximum reaction velocity (Vmax),
and the Michaelis constant (Km). The equation is given as:
V = (Vmax [S])
Km+[S]

Where:
- (V) represents the initial velocity of the reaction.
- Vmax is the maximum velocity or rate of the reaction when the enzyme
is fully saturated with substrate.
- [S] is the substrate concentration.
- Km is the Michaelis constant, which is a measure of the enzyme's
affinity for its substrate. It represents the substrate concentration at
which the reaction velocity is half of Vmax.

Key points about Michaelis-Menten kinetics:

1. Assumptions: The Michaelis-Menten equation is derived based on
several assumptions, including the formation of an enzyme-
substrate complex (ES complex) and the steady-state assumption,
where the rate of formation of ES equals the rate of its breakdown.
2. Initial velocity: the initial velocity of the reaction, is the rate of
product formation at the beginning of the reaction when the
substrate concentration is much higher than the enzyme
concentration.
3. Saturation kinetics: At low substrate concentrations, the rate of the
reaction increases linearly with substrate concentration. However, at
higher substrate concentrations, the enzyme becomes saturated
with substrate, and the rate of the reaction reaches a maximum
(Vmax).
4. Michaelis constant (Km): The Michaelis constant is a measure of
the enzyme's affinity for its substrate. A lower (Km) value indicates
higher affinity, as the enzyme can achieve half of Vmax at lower
substrate concentrations.
 5. Graphical representation: The Michaelis-Menten equation can be
represented graphically using a plot of reaction velocity (V) versus
substrate concentration ([S]). This results in a hyperbolic curve
where the initial velocity approaches (Vmax) as ([S]) increases.
6. Lineweaver-Burk plot: a graphical representation of enzyme
kinetics used to determine Vmax and Km values. It is created by
plotting the reciprocal of reaction velocity ( 1/V) against the
reciprocal of substrate concentration (1/[S]), yielding a straight line
with a slope equal to ( Km / Vmax) and an intercept of 1/ Vmax.

ENZYME INHIBITION
Refers to the decrease in enzyme activity due to the presence of
inhibitors. Inhibitors can be reversible or irreversible In accordance with
the mode of action.

REVERSIBLE INHIBITORS
Reversible inhibitors are a type of enzyme inhibitor that can bind to an
enzyme and temporarily inhibit its activity. Unlike irreversible inhibitors,
which form strong covalent bonds with the enzyme and permanently
deactivate it, reversible inhibitors bind through weaker interactions and
can dissociate from the enzyme, allowing the enzyme to regain its
activity.
Reversible inhibitors, in turn, may be combined in four groups in
accordance with kinetic behavior;

● Competitive inhibitors

● Uncompetitive inhibitors

● Noncompetitive inhibitors

● Mixed inhibitors.

1. Competitive inhibitors bind to the same active site of the enzyme

as the substrate. This prevents the substrate from binding to the
enzyme, and thus slows down the reaction.
2. Non-competitive inhibitors: bind to an allosteric site on the enzyme,
which is distinct from the active site where the substrate binds. The
binding of a non-competitive inhibitor causes a conformational
change in the enzyme, altering its active site's shape and reducing
its catalytic activity.
3. Uncompetitive inhibitors is rather rare, occurring when the inhibitor
is only able to bind to the enzyme once a substrate molecule has
itself bound. As such, inhibition is most significant at high substrate
concentrations, and results in a reduction in the Vmax of the reaction
 4. Mixed inhibitors: also bind to an allosteric site on the enzyme but
can bind whether or not the substrate is already bound to the active
site. However, the affinity of mixed inhibitors for the enzyme can
differ depending on whether the enzyme has already bound to the
substrate or not.

IRREVERSIBLE INHIBITORS
These inhibitors often contain reactive functional groups that modify
amino acid residues of enzyme that are essential for its activity. They also
can provide inhibition affecting the enzyme conformation. An example of
irreversible inhibitor is N-ethylmaleimide that covalently interacts with
SH-group of cysteine residues of enzyme molecules, like peptidase
(insulin-degrading enzyme), 3-phosphoglyceraldehyde dehydrogenase, or
hydrophobic compound from group of cardiotonic steroids that at the last
bind to Na,K-ATPase using hydrophobic interactions. Another well-
known irreversible inhibitor is diisopropyl phosphofluoridate that
modifies OH-group of serine residue in active site of such enzymes as
chymotrypsin and other serine proteases or acetylcholine esterase in
cholinergic synapsis of the nervous system being a potent neurotoxin.

Recommended Reading (https://www.intechopen.com/chapters/54390)

Allosteric Effects
Allosteric effects refer to the regulation of enzyme activity or protein
function through the binding of regulatory molecules at sites other than
the active site. These regulatory sites are known as allosteric sites, and
they can modulate the enzyme's catalytic activity, substrate binding
affinity, or protein conformation. Allosteric regulation plays a crucial role
in cellular processes, metabolic pathways, signal transduction, and
enzyme kinetics.
Allosteric enzymes are key regulatory enzymes that control the activities
of metabolic pathways by responding to inhibitors and activators. These
enzymes in fact show a sigmoidal (S-shaped) relationship between
reaction rate and substrate concentration.
Most allosteric enzymes are polymeric—that is, they are composed of
two or more individual polypeptide chains. They also have multiple active
sites where the substrate can bind. Much of our understanding of the
function of allosteric enzymes comes from studies of hemoglobin.

Allosteric enzymes have an initially low affinity for the substrate, but
when a single substrate molecule binds, this may break some bonds
within the enzyme and thereby change the shape of the protein such that
the remaining active sites are able to bind with a higher affinity.
Therefore allosteric enzymes are often described as moving from
a tensed state or T-state (low affinity) in which no substrate is bound, to
a relaxed state or R-state (high affinity) as substrate binds. Other
molecules can also bind to allosteric enzymes, at additional regulatory
sites (i.e. not at the active site). Molecules that stabilize the protein in its
T-state therefore act as allosteric inhibitors, whereas molecules that move
the protein to its R-state will act as allosteric activators or promoters.
Hemoglobin is a tetrameric protein made up of two alpha subunits and
two beta subunits. It is homologous with the monomeric oxygen-binding
protein, myoglobin. As seen in Figure 6.17, oxygen binding to myoglobin
gives a standard hyperbolic profile, whereas hemoglobin shows sigmoidal
oxygen binding. The sigmoidal kinetic profile of hemoglobin indicates
that oxygen binding is cooperative or that the binding of one oxygen to
one subunit, increases the likelihood that oxygen will bind at another
subunit.

The Concerted Model, also known as the symmetry model, of

hemoglobin is used to explain the cooperativity in oxygen binding as well
as the transitions of proteins which are made up of identical subunits. It
focuses on the two states of the Hemoglobin; the T and R states. The T
state of the hemoglobin is more tense as it is in the deoxyhemoglobin
form while the R state of the hemoglobin is more relaxed as it is in the
oxyhemglobin form. T state is constrained due to the subunit-subunit
interactions while the R state is more flexible due to the ability of oxygen
binding. The difference in deoxyhemoglobin and oxyhemoglobin is that
the “deoxy” form doesn’t have oxygen and the “oxy” form is highly
oxygen bound. The binding of oxygen at one site increases or facilitates
the binding affinity in other active sites. Thus, a sigmoidal curve is
observed for the oxygen binding kinetics of hemoglobin. As seen in
Figure 6.18, in the concerted model of the hemoglobin, it shows that the
one oxygen binding to an active site will increase the probability of other
oxygen binding to the other active sites in the hemoglobin. This ability is
known as cooperativity or cooperative binding. Overall, oxygen binding
shifts the equilibrium toward the R state. This means that at high oxygen
levels, the R form will be prevalent and at lower oxygen levels, the T
form will be prevalent. Allosteric effectors of hemoglobin, such as 2,3-
bisphosphoglycerate (2,3-BPG), function by shifting the equilibrium
towards or away from the T-state, depending on whether it’s an allosteric
inhibitor or allosteric promoter. This model displays the extreme cases of
R and T transitions. In a real system, properties from both models are
needed to explain the behavior of hemoglobin.

Recommended reading
(https://wou.edu/chemistry/courses/online-chemistry-textbooks/ch450-
and-ch451-biochemistry-defining-life-at-the-molecular-level/chapter-7-
enzyme-kinetics/)
Enzyme assay in clinical medicine.

An enzyme assay is a laboratory technique used to measure the activity,

concentration, or properties of enzymes in biological samples. These
techniques are used to quantitatively or qualitatively measure enzymes'
activity, concentration, or other properties in biological samples.

They are crucial in biochemistry, molecular biology, pharmacology, and

clinical medicine for studying enzyme kinetics, diagnosing diseases,
monitoring treatments, and evaluating enzymatic processes.

Types of Enzyme Assays

1. Activity-Based Assays
- Measure the catalytic activity of enzymes, often involving the
conversion of substrates into products.
 - Examples include measuring the rate of product formation or
substrate consumption in an enzymatic reaction.

2. Concentration-Based Assays
- Quantify the concentration of enzymes in a sample, typically
expressed as units of enzyme activity per volume of sample (e.g., units
per milliliter).
- Examples include enzyme-linked immunosorbent assays (ELISA) for
measuring enzyme concentration using specific antibodies.

3. Property-Based Assays
- Evaluate specific properties of enzymes, such as stability, substrate
specificity, cofactor dependence, or inhibition.
- Examples include thermal stability assays, substrate specificity assays,
and inhibition assays.

Methods Used in Enzyme Assays

1. Spectrophotometric Assays
- Measure enzyme activity based on changes in absorbance or emission
of light caused by enzymatic reactions.
- Examples include monitoring color changes, UV absorption, or
fluorescence in reaction mixtures.
 - Common spectrophotometric assays include the Bradford assay for
protein concentration and the lactate dehydrogenase (LDH) assay for
enzyme activity.

2. Chromatographic Assays
- Separate and quantify enzyme substrates, products, or reaction
components using chromatography techniques such as high-performance
liquid chromatography (HPLC) or gas chromatography (GC).
- Chromatographic assays are useful for studying enzyme kinetics and
identifying reaction intermediates.

3. Electrophoretic Assays
- Separate enzymes based on their charge, size, or other properties
using electrophoresis techniques such as gel electrophoresis or capillary
electrophoresis.
- Enzymes can be visualized, quantified, or characterized based on their
migration patterns in electrophoretic gels.

4. Immunoassays
- Utilize antibodies to detect and quantify enzymes or enzyme-
substrate complexes.
 - ELISA (enzyme-linked immunosorbent assay) is a widely used
immunoassay technique for measuring enzyme concentration in
biological samples.

5. Radioactive Assays
- Use radioactive isotopes to label substrates, products, or reaction
components, allowing for sensitive detection and quantification of
enzyme activity.
- Radioactive assays are particularly useful for studying metabolic
pathways and enzyme kinetics.

6. Molecular Biology Assays

- Employ molecular biology techniques such as polymerase chain
reaction (PCR), cloning, sequencing, and gene expression analysis to
study enzymes at the genetic and molecular level.
- Molecular biology assays are essential for studying enzyme structure,
function, regulation, and genetic variations.

Considerations in Enzyme Assays

- Specificity: Ensuring that the assay measures the activity or
concentration of the target enzyme accurately without interference from
other enzymes or compounds.
- Sensitivity: Detecting low levels of enzyme activity or concentration,
especially in clinical or research settings where enzyme levels may be low.
- Linearity: Ensuring that the assay response (e.g., absorbance,
fluorescence) is proportional to the enzyme activity or concentration over
a range of values.
- Reproducibility: Achieving consistent and reproducible results across
different experimental conditions, replicates, and laboratories.
- Validation: Validating the assay's accuracy, precision, reliability, and
robustness through calibration, quality control measures, and comparison
with reference methods or standards.