INTRODUCTION

Microbial biochemistry comprises of biochemical reactions in microbial growth, various modes

and mechanisms/ processes of pathogenesis required in causing infection/ diseases in the host. It
involves the study of microbial growth, microbial cell structure, microbial metabolism, primary
and advanced functions and the interactions of biological macromolecules, like carbohydrates,
proteins, Fatty Acids and Lipids and nucleic acids; which cater the skeletal aspect and basis of
functions affiliated with life. Biochemical study of microbes is crucial in the processes of their
action. Post genomic analyses, maintenance of mechanisms, & functional replication, integrating
plasmid functions, conjugation systems and regulatory network are the key factors that play a
vital role in metabolism of microbes. When monomers are co-linked to synthesize a polymer,
dehydration occurs often resulting in assembly of different macromolecules in a much larger
complexes.
 Chemical Biology
 Chemical Microbiology
 Microbial metabolism
 Microbial Genetics
 Microbial Assay
 Biochemistry is the discipline that studies the chemistry of life, and its objective is to
explain form and function based on chemical principles. Organic chemistry is the
discipline devoted to the study of carbon-based chemistry, which is the foundation for the
study of biomolecules and the discipline of biochemistry.

ELEMENTS IN LIVING CELLS: MAJOR AND MINOR ESSENTIAL ELEMENTS

The most abundant element in cells is hydrogen (H), followed by carbon (C), oxygen (O),
nitrogen (N), phosphorous (P), and sulfur (S). We call these elements macronutrients, and they
account for about 99% of the dry weight of cells. Some elements, such as sodium (Na),
potassium (K), magnesium (Mg), zinc (Zn), iron (Fe), calcium (Ca), molybdenum (Mo), copper
(Cu), cobalt (Co), manganese (Mn), or vanadium (Va), are required by some cells in very small
amounts and are called micronutrients or trace elements.
All of these elements are essential to the function of many biochemical reactions, and, therefore,
are essential to life.
The four most abundant elements in living matter (C, N, O, and H) have low atomic numbers and
are thus light elements capable of forming strong bonds with other atoms to produce molecules.
Carbon forms four chemical bonds, whereas nitrogen forms three, oxygen forms two, and
hydrogen forms one. When bonded together within molecules, oxygen, sulfur, and nitrogen often
have one or more “lone pairs” of electrons that play important roles in determining many of the
molecules’ physical and chemical properties.
These traits in combination permit the formation of a vast number of diverse molecular species
necessary to form the structures and enable the functions of living organisms.

Organic molecules contain carbon; inorganic compounds do not. Carbon oxides and carbonates
are exceptions; they contain carbon but are considered inorganic because they do not contain
hydrogen. The atoms of an organic molecule are typically organized around chains of carbon
atoms.
Most of the carbon found in organic molecules originates from inorganic carbon sources such as
carbon dioxide captured via carbon fixation by microorganisms.
Organic molecules in organisms are generally larger and more complex than inorganic
molecules. Their carbon skeletons are held together by covalent bonds. They form the cells of an
organism and perform the chemical reactions that facilitate life. All of these molecules, called
biomolecules because they are part of living matter, contain carbon, which is the building block
of life.
Carbon is a very unique element in that it has four valence electrons in its outer orbitals and can
form four single covalent bonds with up to four other atoms at the same time.
These atoms are usually oxygen, hydrogen, nitrogen, sulfur, phosphorous, and carbon itself; the
simplest organic compound is methane, in which carbon binds only to hydrogen.

In addition to containing carbon atoms, biomolecules also contain functional groups—groups of

atoms within molecules that are categorized by their specific chemical composition and the
chemical reactions they perform, regardless of the molecule in which the group is found.
Inorganic compounds make up 1%–1.5% of a living cell’s mass. Living organisms contain
inorganic compounds (mainly water and salts; and organic molecules. They are small, simple
compounds that play important roles in the cell, although they do not form cell structures.

WATER
Water is the most abundant substance in living systems, making up 70% or more of the weight of
most organisms.
Water is both the solvent in which metabolic reactions occur and a reactant in many biochemical
processes, including hydrolysis, condensation, and oxidation-reduction reactions.
Water has a higher melting point, boiling point, and heat of vaporization than most other
common solvents. These unusual properties are a consequence of liquid water great internal
cohesion. A look at the electron structure of the H 2O molecule reveals the cause of these
intermolecular attractions.
Close water molecules are attracted to each other by a relatively low electrical attraction,
(negative hydrogen atoms attract positive oxygen atoms in other molecules). This bond is called
a ‘hydrogen bond’. Water has unique properties because of its polarity and the hydrogen bonds
between its molecules.
Physical properties of water
 The molecule of water has covalent bonding between Hydrogen and Oxygen atoms. Two
hydrogen atoms form a bond with a single atom of oxygen.
 Appearance: Water is colorless, odorless and tasteless liquid in its natural state.
 Boiling Point: As we know, water has a boiling point of 100 C.
Freezing Point: The same concept applies to the freezing point of water as well. The freezing
point of water is 0 C.
Density: One unique property of water is that in the sold state, it is lense dense. Up to 4°C
water’s density does increase on cooling. But after that point water becomes less dense. This
is why ice floats in water,
 Viscosity: Water has low viscosity.
 Solvency: Water is an excellent solvent. In fact, it is known as a Universal Solvent. Due to a
water molecule’s polarity, it can dissolve almost any substance.
Water is a polar solvent.
 Water is regarded as the ‘general solvent’ or ‘universal solvent’ due to the polarity of its
molecules.
 For example, when sodium chloride (NaCl) dissolves in water, it produces positive
sodium ions and negative chlorine ions. The positive oxygen atoms in water attract the
negative chlorine ions, and the negative hydrogen atoms attract the positive sodium ions.
All polar substances (substances containing ions) can dissolve in polar solvents, such as
water.
 All the essential substances for living organisms (vitamins, salts, amino acids, gases, and
glucose) transport inside their bodies in the form of solutes dissolved in water. These
substances take part in metabolic reactions inside the cells.
2- Water has the ability to ionize molecules, which are necessary for life.
 This means that water has the ability to disassociate the molecules necessary for life into
positive and negative ions (water can do so due to the polarity of its molecules).
For example, the pancreas secretes sodium bicarbonate (NaHCO3). This compound
ionizes in water into positive hydrogen ions and negative bicarbonate ions, which makes
the medium alkaline and thus suitable for the enzymes’ work.
3- Water has high specific heat.
 Specific heat is the amount of heat required to increase the temperature of one gram of
matter by 1 degree Celsius.
 Water has the highest specific heat on Earth due to the hydrogen bonds between its
molecules.
 As a result of having high specific heat, water needs a great amount of energy to increase
its temperature and loses a great amount of energy when its temperature decreases. This
helps living organisms to have a constant temperature which is essential for the vital
processes occurring within their bodies. Cells contain lots of water to keep their
temperature constant.
 Animals and plants lose water by sweating and transpiration processes to decrease their
temperature.
 The high specific heat of water provides living organisms with temperatures suitable for
life on Earth.
 Water forms almost 70% of the surface area of Earth. If water didn’t exist in such a great
amount, the temperature of the Earth would decrease dramatically because the substances
forming the Earth’s crust have low specific heat.
 The water that makes up oceans absorb a great number of sun rays in the morning and
spread them into the atmosphere at night in order to keep the temperature of the Earth
suitable for living organisms.
4- Water has low viscosity and high surface tension.
 Surface tension is the cohesion of the molecules on the surface of a fluid to occupy the
least possible volume. Viscosity is the resistance of a fluid to flowing.
 Water has low viscosity and high surface tension due to the hydrogen bonds between its
molecules; these conditions are suitable for life.
These properties are important because:
1- They work on the cohesion of cell substances.
2- It slows down water loss in plants’ leaves through pores.
3- Some insects can walk on water due to the cohesion of the molecules on its surface.
5- Water density decreases under 4◦C.
 Water expands when its temperature becomes less than 4◦C (instead of shrinking). THis
decreases its density and makes it float. In frozen lakes, we find ice on the surface, while
we find liquid water underneath.
 This property is because of the hydrogen bonds between water molecules.
 This property is important because it enables living organisms to live in oceans and seas.
Without this property, all oceans and seas will turn into ice, rather than just the surface.
Surface freezing works as an insulator to prevent the rest of water from freezing.
6- The freezing point of water decreases if it has substances dissolved in it.
 This property is very important for living organisms, as it prevents the water in the cells
of from freezing when exposed to temperatures less than 0◦C.
7- Water can turn into vapour in temperatures lower than boiling point (100◦C).
 Water vapour formed on the surfaces of oceans is carried by convection currents to cold
layers in the atmosphere. This changes into clouds which provide living organisms with
rain and water.
8- Water rise in capillary tubes.
 Water has the ability to rise in capillary tubes without being pumped and in opposition to
external forces such as gravity. This property helps water transport from trees’ roots to all
of its parts.

Chemical properties of water

 Amphoteric Nature: An amphoteric substance is one which can act as an acid or a base. While
 Water is neither acidic or basic, it has neutral Ph of 7.
 It has a strong hydrating tendency. Water has strong reactions with ions of salts and creates
hydrating shells around them.
SiCl4 + 2H2O → SiO2 + 4HCl
 Redox Reactions: Water is a great source to obtain dihydrogen since it can be reduced by
reacting it with highly electropositive metals such as Sodium.
H2O + Na → 2NaOH + H2
The attractive forces between water molecules and the slight tendency of water to ionize are of
crucial importance to the structure and function of biomolecules.
The water molecule and its ionization products, H_ and OH_, profoundly influence the structure,
self-assembly, and properties of all cellular components, including proteins, nucleic acids, and
lipids.
The noncovalent interactions responsible for the strength and specificity of “recognition” among
biomolecules are decisively influenced by the solvent properties of water, including its ability to
form hydrogen bonds with itself and with solutes.

Hydrogen bonds between water molecules provide the cohesive forces that make water a liquid
at room temperature and that favor the extreme ordering of molecules that is typical of
crystalline water (ice).
Polar biomolecules dissolve readily in water while, nonpolar biomolecules interfere with water-
water interactions and are unable to form water-solute interaction.
Hydrogen bonds and ionic, hydrophobic (Greek, “water-fearing”), and van der Waals
interactions are individually weak, but collectively they have a very significant influence on the
three-dimensional structures of proteins, nucleic acids, polysaccharides, and membrane
lipids.
Each hydrogen atom of a water molecule shares an electron pair with the central oxygen atom.
As a result, there is an electrostatic attraction between the oxygen atom of one water molecule
and the hydrogen of another, called a hydrogen bond.
Hydrogen bonds are relatively weak. Those in liquid water have a bond dissociation energy (the
energy required to break a bond).
It readily dissolves most biomolecules, which are generally charged or polar compounds;
compounds that dissolve easily in water are hydrophilic (Greek, “water-loving”).
In contrast, nonpolar solvents such as chloroform and benzene are poor solvents for polar
biomolecules but easily dissolve those that are hydrophobic—nonpolar molecules such as lipids
and waxes.

The value of 7 for the pH of a precisely neutral solution is not an arbitrarily chosen figure; it is
derived from the absolute value of the ion product of water at 25 _C, which by convenient
coincidence is a round number.
Solutions having a pH greater than 7 are alkaline or basic; the concentration of OH_ is greater
than that of H_. Conversely, solutions having a pH less than 7 are acidic.
Water is not just the solvent in which the chemical reactions of living cells occur; it is very often
a direct participant in those reactions. The formation of ATP from ADP and inorganic phosphate
is an example of a condensation reaction in which the elements of water are eliminate.

Many biomolecules are amphipathic; proteins, pigments, certain vitamins, and the sterols and
phospholipids of membranes all have polar and nonpolar surface regions. Structures composed
of these molecules are stabilized by hydrophobic interactions among the nonpolar
regions. Hydrophobic interactions among lipids, and between lipids and proteins, are the most
important determinants of structure in biological membranes.
Hydrophobic interactions between nonpolar amino acids also stabilize the three-dimensional
structures of proteins.

Although many of the solvent properties of water can be explained in terms of the uncharged
H2O molecule, the small degree of ionization of water to hydrogen ions (H_) and hydroxide ions
(OH_) must also be taken into account.
When weak acids are dissolved in water, they contribute H_ by ionizing; weak bases consume
H_ by becoming protonated.

Water molecules have a slight tendency to undergo reversible ionization to yield a hydrogen ion
(a proton) and a hydroxide ion, giving the equilibrium
ORGANIC COMPOUNDS
Compounds which are obtained from plants and animals are organic compounds and compounds
which are obtained from minerals, non-living sources are termed as inorganic compounds.
However, the modern definition of organic compounds is a bit different to this.
An organic compound
is defined as any compound whose molecules contain carbon and hydrogen ( also known as ”
hydrocarbons” ) or compound that is the derivative of it. The branch of science which deals with the
scientific study of structure, properties and reactions of hydrocarbons and their derivatives is known
as organic chemistry.
The general characteristics of Organic Compounds include:
 Can be isolated as well as prepared in laboratory
 Comprise almost 90% of all known compounds.
 Mostly built up of only three elements- carbon, hydrogen and oxygen. Other elements like
halogen, nitrogen as well as phosphorous are also present but to a lesser extent.
 Possess complex structures and high molecular weights
 Their properties are decided by certain active atom or group of atoms known as the functional
group.
 They are mostly insoluble in water but soluble in organic solvents.
 They are combustible in nature
 Chemical reactions involving organic compounds proceed at slower rates.

A covalent bond is a chemical bond that involves the sharing of electron pairs between atoms that in
turn results in a balance of attractive and repulsive forces between the atoms. The presence of a
covalent bond renders certain characteristics to the organic compounds. These include:
 Low melting points and boiling points in comparison to the inorganic compounds.
 Organic acids and bases are less stronger and thus they have a limited dissociation in an
aqueous medium.
 They exhibit the phenomenon of isomerism in which a single molecular formula represents
several organic compounds differing in physical and chemical properties.
 They are volatile in nature.
General Characteristics of Members of Homologous Series
A Homologous Series is a group of organic chemical compounds, usually listed in the order of
increasing size, that have a similar structure (and hence, also similar properties) and whose
structures differ only by the number of CH2– CH2 units in the main carbon chain. They possess the
following general characteristics:
 A general formula describes the members of the homologous series
 Successive members differ from each other by CH2CH2
 Physical properties change regularly with increasing number of carbon atoms.
 Members have similar chemical properties because they have same functional group.
 Members of the homologous series can be prepared using the same method.
Importance of Organic Compounds
 Organic compounds are important because all living organisms contain carbon.
 While carbohydrates, proteins and fats, the basic structures of life, are organic compounds
 They are the basic components of many of the cycles that drive the earth. For example, the
`carbon cycle that includes the exchange of carbon between plants and animals in
photosynthesis and cellular respiration.
 Organic compounds combine with metals to form organometallic compounds. These
compounds are important industrially. They are used as catalysts, promoters, analysers as well
as stabilizers.

Nutritional Requirements of Microorganisms

Classification by Carbon and Energy Source
Organisms can be identified according to the source of carbon they use for metabolism as well as
their energy source. Organisms that convert inorganic carbon dioxide (CO 2) into organic carbon
compounds are autotrophs.
Conversely, heterotrophs rely on more complex organic carbon compounds as nutrients; these
are provided to them initially by autotrophs.
Those that get their energy for electron transfer from light are phototrophs,
whereas chemotrophs obtain energy for electron transfer by breaking chemical bonds.
There are two types of chemotrophs: organotrophs and lithotrophs.
Organotrophs obtain energy from organic compounds.
Lithotrophs (“litho” means “rock”) are chemotrophs that get energy from inorganic compounds,
including hydrogen sulfide (H2S) and reduced iron. Lithotrophy is unique to the microbial world.
Most organisms are chemoheterotrophs because they use organic molecules as both their
electron and carbon sources.

Classifications of Organisms by Energy and Carbon Source

Energy Carbon
Classifications Examples
Source Source

Hydrogen-, sulfur-, iron-, nitrogen-,

Chemoautotrophs Chemical Inorganic and carbon monoxide-oxidizing
Chemotroph bacteria
s
Organic All animals, most fungi, protozoa, and
Chemoheterotrophs Chemical
compounds bacteria

All plants, algae, cyanobacteria, and

Photoautotrophs Light Inorganic
green and purple sulfur bacteria
Phototrophs
Organic Green and purple nonsulfur bacteria,
Photoheterotrophs Light
compounds heliobacteria

Mineral Nutrients:
The microbial nutrients can be classified as macro (major) nutrients, and micro (minor) nutrients
or trace elements on the basis of their amount required.
1. Macro or Major Mineral Nutrients:
The microbial cells contain water accounting for some 80-90% of their total weight and,
therefore, the water is always the major essential nutrient in quantitative terms.
The solid matter of cells contain, in addition to oxygen and hydrogen (derivable metabolically
from water), the other macro (major) elements, namely, carbon, nitrogen, phosphorus, sulphur,
potassium, magnesium, sodium, calcium and iron in order of decreasing abundance.
About 95% of cellular dry weight of microbial cells is accounted for only six macro (major)
elements (O, H, C, N, P and S).
Carbon assumes great importance as the main constituent of all organic cell materials and
represents about 50% of cell’s dry weight. CO2 is the most oxidized form of carbon and the
photo-synthetic microorganisms reduce CO2 to organic cell constituents. On the other hand, all
the non-photosynthetic microorganisms obtain their carbon requirement mainly from organic
nutrients which contain reduced carbon compounds.
These organic compounds not only provide the carbon for synthesis but also meet the energy
requirement by entering into energy yielding metabolic pathways and are eventually oxidised to
CO2.
Some microbes have the ability to synthesize all their cellular components using a single organic
carbon source while others, in addition to this one major carbon source, also need other complex
carbon containing components which they cannot synthesize.
These components are called growth factors and include vitamins. Some microbes can utilize
more than one carbon compound and exhibit a great degree of versatility. The others, however,
are specialized in this regard.
Sulphur and nitrogen are taken up by most organisms and are subsequently reduced within the
cell and utilized in other biosynthetic processes.
The sulphur and nitrogen requirements of most organisms can also be met with organic nutrients
that contain these two elements in reduced organic combinations such as amino acids.
A few microorganisms are capable of reducing elemental nitrogen to ammonia and this process
of nitrogen assimilation is known as biological nitrogen fixation.
Oxygen is a component of the cellular material of all the microorganisms. Most of the
microorganisms need molecular oxygen for respiration. In these, the oxygen serves as terminal
electron acceptor, and such organisms are referred to as ‘obligate aerobes’.
As opposed to this there are a few organisms which do not use molecular oxygen as terminal
electron acceptor. These microbes are called ‘obligate anaerobes’. In fact, molecular oxygen is
toxic to these organisms.
Aerobes which can grow in the absence of oxygen are called ‘facultative anaerobes’ and the
anaerobes which can grow in the presence of oxygen are referred to as ‘facultative aerobes’. In
addition to these major classes, there are organisms which grow best at reduced oxygen pressure
but are obligate aerobes and these are called ‘Microaerophilic’.
Micro or Minor Mineral Nutrients or Trace Elements:
The microorganisms, in general do not use only macro (major) elements but also others like
cobalt, copper, manganese, molybdenum, nickel, selenium, tungsten, vanadium and zinc which
are required in residual fraction by nearly all microorganisms.
These elements are often referred to as minor (micro) nutrients or trace elements. The
micronutrients or trace elements are nevertheless just as critical to cell function as are the
macronutrients.
They are metals playing the role of cell’s catalysts and many of them are play a structural role in
various enzymes.
Some microorganisms, however, need additional specific mineral nutrients, for example, diatoms
and some microalgae require silica, supplied as silicate, to impregnate their cell walls.
Growth Factors:
Besides the mineral nutrients, the microorganisms need some organic compounds. Most of the
microorganisms are capable of synthesizing these organic compounds from simpler carbon
resources, others cannot and need their supply from outside for their proper growth and
development.
Organic nutrients of this type are known collectively as growth factors (essential metabolites)
and can be categorized into three groups (amino acids, purines and pyrimidines and vitamins) on
the basis of their chemical structure and metabolic function.
Amino acids and purines and pyrimidines are the constituents of proteins and nucleic acids,
respectively. Vitamins, however, are the most commonly needed growth factor and form parts of
the prosthetic groups or active centres of certain enzymes.

Organic nutrients of this type are known collectively as growth factors (essential metabolites)
and can be categorized into three groups (amino acids, purines and pyrimidines and vitamins) on
the basis of their chemical structure and metabolic function.
Amino acids and purines and pyrimidines are the constituents of proteins and nucleic acids,
respectively. Vitamins, however, are the most commonly needed growth factor and form parts of
the prosthetic groups or active centres of certain enzymes.

Since the growth factors fulfill specific needs in biosynthesis of certain molecules, they are
needed in very small amounts; the vitamins even in less smaller quantities, because of the
various coenzymes of which they are precursors, have catalytic roles and consequently are
present at levels of a few parts per million in the microbial cell.

Some important vitamins and their functions are summarized in Table 18.3.
Some important vitamins and their functions are summarized in Table 18.3.

MICROBIAL METABOLISM
Metabolism refers to all of the chemical reactions inside a cell.
By metabolizing such substances, microbes chemically convert them to other forms. In some
cases, microbial metabolism produces chemicals that can be harmful to other organisms; in
others, it produces substances that are essential to the metabolism and survival of other life
forms.
Prokaryotes have great metabolic diversity with important consequences to other forms of life.
Cellular processes such as the building or breaking down of complex molecules occur through
series of stepwise, interconnected chemical reactions called metabolic pathways. Reactions that
are spontaneous and release energy are exergonic reactions.
Reactions that require energy /fueled by the use of cellular energy are endergonic.
The term anabolism refers to those endergonic metabolic pathways involved in converting
simple molecular building blocks into more complex molecules.
Catabolism refers to exergonic pathways that break down complex molecules into simpler ones.
Molecular energy stored in complex molecules is released in catabolic pathways and harvested to
drive anabolic pathways. Thus, in terms of energy and molecules, cells are continually balancing
catabolism with anabolism.
Oxidation and Reduction in Metabolism
Transfer of electrons between molecules is important because most of the energy stored in
atoms and used to fuel cell functions is in the form of high-energy electrons.
The transfer of energy in the form of electrons allows the cell to transfer and use energy
incrementally; that is, in small packages rather than a single, destructive burst.
Reactions that remove electrons from donor molecules, leaving them oxidized, are oxidation
reactions.
Reduction reactions add electrons to acceptor molecules, leaving them reduced.
Because electrons can move from one molecule to another, oxidation and reduction occur in
tandem.
These pairs of reactions are called oxidation-reduction reactions or redox reactions.

Energy Carriers: NAD+, NADP+, FAD, and ATP

Energy released from the breakdown of the chemical bonds within nutrients can be stored either
through the reduction of electron carriers or in the bonds of adenosine triphosphate (ATP).
A living cell must be able to handle the energy released during catabolism in a way that enables
the cell to store energy safely and release it for use only as needed.
Living cells accomplish this by using the compound adenosine triphosphate (ATP).

In living systems, a small class of compounds functions as mobile electron carriers. These

molecules bind to and shuttle high-energy electrons between compounds in pathways.
Principal electron carriers originate from B vitamin group and are derivatives of nucleotide. They
include: Nicotinamide adenine dinucleotide (NAD+/NADH), Nicotine adenine dinucleotide
phosphate (NADP+), and Flavin adenine dinucleotide (FAD).
These compounds can be easily reduced or oxidized.
Nicotinamide adenine dinucleotide (NAD+/NADH) is the most common mobile electron carrier
used in catabolism.
NAD+ is the oxidized form of the molecule; NADH is the reduced form of the molecule.
Nicotine adenine dinucleotide phosphate (NADP+), the oxidized form of an NAD + variant that
contains an extra phosphate group, is another important electron carrier; it forms NADPH when
reduced.
The oxidized form of flavin adenine dinucleotide is FAD, and its reduced form is FADH2.

Collectively, FADH2, NADH, and NADPH are often referred to as having reducing power due to
their ability to donate electrons to various chemical reactions.

ATP is often called the “energy currency” of the cell, and, like currency, this versatile compound
can be used to fill any energy need of the cell.

At the heart of ATP is a molecule of adenosine monophosphate (AMP), which is composed of

an adenine molecule bonded to a ribose molecule and a single phosphate group.
Ribose is a five-carbon sugar found in RNA, and AMP is one of the nucleotides in RNA.
The addition of a second phosphate group to this core molecule results in the formation of
adenosine diphosphate (ADP); the addition of a third phosphate group forms ATP.
Adding a phosphate group to a molecule, a process called phosphorylation, requires energy.

Enzyme Structure and Function

Enzymes thus play an important role in controlling cellular metabolism. Enzymes are inorganic
molecules that serve as catalysts for a wide range of chemical reactions inside cells.

A catalyst is a substance that helps speed up a chemical reaction. Catalysts are not used or
changed during chemical reactions and, therefore, are reusable
An enzyme functions by lowering the activation energy of a chemical reaction inside the cell.
Activation energy is the energy needed to form or break chemical bonds and convert reactants to
products.
Enzymes lower the activation energy by binding to the reactant molecules and holding them in
such a way as to speed up the reaction.
The chemical reactants to which an enzyme binds are called substrates.
Due to a jigsaw puzzle-like match between an enzyme and its substrates, enzymes are known for
their specificity.

Factor that influences enzyme activity

Enzymes are subject to influences by local environmental conditions such as:
Temperature
Although increasing the environmental temperature generally increases reaction rates, enzyme
catalyzed or otherwise, increasing or decreasing the temperature outside of an optimal range can
affect chemical bonds within the active site, making them less well suited to bind substrates.
High temperatures will eventually cause enzymes, like other biological molecules, to denature,
losing their three-dimensional structure and function. Microbes that inhabit hot springs have
enzymes that work best at high temperatures, human pathogens have enzymes that work best at
37°C.

pH
Enzymes are also suited to function best within a certain pH range, and, as with temperature,
extreme environmental pH values (acidic or basic) can cause enzymes to denature. Similarly,
while enzymes produced by most organisms work best at a neutral pH, microbes growing in
acidic environments make enzymes optimized to low pH conditions, allowing for their growth at
those conditions.

Substrate concentration:
Enzyme activity is increased at higher concentrations of substrate until it reaches a saturation
point at which the enzyme can bind no additional substrate.
Overall, enzymes are optimized to work best under the environmental conditions in which the
organisms that produce them live.

Many enzymes do not work optimally, or even at all, unless bound to other specific nonprotein
helper molecules, either temporarily through ionic or hydrogen bonds or permanently through
stronger covalent bonds.
Binding to these molecules promotes optimal conformation and function for their respective
enzymes.
Two types of helper molecules are cofactors and coenzymes.
Cofactors are inorganic ions such as iron (Fe 2+) and magnesium (Mg2+) that help stabilize
enzyme conformation and function. One example of an enzyme that requires a metal ion as a
cofactor is the enzyme that builds DNA molecules, DNA polymerase, which requires a bound
zinc ion (Zn2+) to function.
Coenzymes are organic helper molecules that are required for enzyme action.
Like enzymes, they are not consumed and, hence, are reusable.

Some cofactors and coenzymes like often bind to the enzyme’s active site, aiding in the
chemistry of the transition of a substrate to a product.
In such cases, an enzyme lacking a necessary cofactor or coenzyme is called an apoenzyme and
is inactive.
Conversely, an enzyme with the necessary associated cofactor or coenzyme is called a
holoenzyme and is active.
NADH and ATP are also both examples of commonly used coenzymes that provide high-energy
electrons or phosphate groups respectively, which bind to enzymes, thereby activating them.

Enzyme Inhibitors
There are many different kinds of molecules that inhibit or promote enzyme function, and
various mechanisms exist for doing so. Enzymes can be regulated in ways that either promote or
reduce their activity.
A competitive inhibitor is a molecule similar enough to a substrate that it can compete with the
substrate for binding to the active site by simply blocking the substrate from binding.
For a competitive inhibitor to be effective, the inhibitor concentration needs to be approximately
equal to the substrate concentration.

Sulfa drugs provide a good example of competitive competition. They are used to treat bacterial
infections because they bind to the active site of an enzyme within the bacterial folic acid
synthesis pathway.

When present in a sufficient dose, a sulfa drug prevents folic acid synthesis, and bacteria are
unable to grow because they cannot synthesize DNA, RNA, and proteins.

A noncompetitive (allosteric) inhibitor binds to the enzyme at an allosteric site, a location

other than the active site, and still manages to block substrate binding to the active site by
inducing a conformational change that reduces the affinity of the enzyme for its substrate.
Because only one inhibitor molecule is needed per enzyme for effective inhibition, the
concentration of inhibitors needed for noncompetitive inhibition is typically much lower than the
substrate concentration.
In addition to allosteric inhibitors, there are allosteric activators that bind to locations on an
enzyme away from the active site, inducing a conformational change that increases the affinity of
the enzyme’s active site(s) for its substrate(s).
Allosteric control is an important mechanism of regulation of metabolic pathways involved in
both catabolism and anabolism.

In a most efficient and elegant way, cells have evolved also to use the products of their own
metabolic reactions for feedback inhibition of enzyme activity.
Feedback inhibition involves the use of a pathway product to regulate its own further
production. The cell responds to the abundance of specific products by slowing production
during anabolic or catabolic reactions.
Glycolysis
Glycolysis, which translates to "splitting sugars", is the process of releasing energy within
sugars. In glycolysis, a six-carbon sugar known as glucose is split into two molecules of a three-
carbon sugar called pyruvate. This multistep process yields two ATP molecules containing free
energy, two pyruvate molecules, two high energy, electron-carrying molecules of NADH, and
two molecules of water.
The ATP molecules produced during the energy payoff phase of glycolysis are formed by
substrate-level phosphorylation, one of two mechanisms for producing ATP. In substrate-level
phosphorylation, a phosphate group is removed from an organic molecule and is directly
transferred to an available ADP molecule, producing ATP.
During glycolysis, high-energy phosphate groups from the intermediate molecules are added to
ADP to make ATP.

Glycolysis

 Glycolysis is the process of breaking down glucose.

 Glycolysis can take place with or without oxygen.
 Glycolysis produces two molecules of pyruvate, two molecules of ATP, two molecules
of NADH, and two molecules of water.
 Glycolysis takes place in the cytoplasm.
 There are 10 enzymes involved in breaking down sugar. The 10 steps of glycolysis are
organized by the order in which specific enzymes act upon the system.
Glycolysis can occur with or without oxygen. In the presence of oxygen, glycolysis is the first
stage of cellular respiration. In the absence of oxygen, glycolysis allows cells to make small
amounts of ATP through a process of fermentation.
Glycolysis takes place in the cytosol of the cell's cytoplasm. A net of two ATP molecules are
produced through glycolysis (two are used during the process and four are produced.) Learn
more about the 10 steps of glycolysis below.
Step 1
The enzyme hexokinase phosphorylates or adds a phosphate group to glucose in a
cell's cytoplasm. In the process, a phosphate group from ATP is transferred to glucose
producing glucose 6-phosphate or G6P. One molecule of ATP is consumed during this phase.
Step 2
The enzyme phosphoglucomutase isomerizes G6P into its isomer fructose 6-phosphate or F6P.
Isomers have the same molecular formula as each other but different atomic arrangements.
Step 3
The kinase phosphofructokinase uses another ATP molecule to transfer a phosphate group to
F6P in order to form fructose 1,6-bisphosphate or FBP. Two ATP molecules have been used so
far.
Step 4
The enzyme aldolase splits fructose 1,6-bisphosphate into a ketone and an aldehyde molecule.
These sugars, dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP), are
isomers of each other.
Step 5
The enzyme triose-phosphate isomerase rapidly converts DHAP into GAP (these isomers can
inter-convert). GAP is the substrate needed for the next step of glycolysis.
Step 6
The enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves two functions in
this reaction. First, it dehydrogenates GAP by transferring one of its hydrogen (H⁺) molecules to
the oxidizing agent nicotinamide adenine dinucleotide (NAD⁺) to form NADH + H⁺.

Next, GAPDH adds a phosphate from the cytosol to the oxidized GAP to form 1,3-
bisphosphoglycerate (BPG). Both molecules of GAP produced in the previous step undergo this
process of dehydrogenation and phosphorylation.

Step 7
The enzyme phosphoglycerokinase transfers a phosphate from BPG to a molecule of ADP to
form ATP. This happens to each molecule of BPG. This reaction yields two 3-phosphoglycerate
(3 PGA) molecules and two ATP molecules.
Step 8
The enzyme phosphoglyceromutase relocates the P of the two 3 PGA molecules from the third
to the second carbon to form two 2-phosphoglycerate (2 PGA) molecules.
Step 9
The enzyme enolase removes a molecule of water from 2-phosphoglycerate to form
phosphoenolpyruvate (PEP). This happens for each molecule of 2 PGA from Step 8.
Step 10
The enzyme pyruvate kinase transfers a P from PEP to ADP to form pyruvate and ATP. This
happens for each molecule of PEP. This reaction yields two molecules of pyruvate and two ATP
molecules.
Cite this Article 

For bacteria, eukaryotes, and most archaea, glycolysis is the most common pathway for the
catabolism of glucose; it produces energy, reduced electron carriers, and precursor molecules for
cellular metabolism. Every living organism carries out some form of glycolysis, suggesting this
mechanism is an ancient universal metabolic process. The process itself does not use oxygen;
however, glycolysis can be coupled with additional metabolic processes that are either aerobic or
anaerobic.
Glycolysis takes place in the cytoplasm of prokaryotic and eukaryotic cells. It begins with a
single six-carbon glucose molecule and ends with two molecules of a three-carbon sugar called
pyruvate.

Other Glycolytic Pathways

The type of glycolysis found in animals and that is most common in microbes is the Embden-
Meyerhof-Parnas (EMP) pathway, named after Gustav Embden (1874–1933), Otto Meyerhof
(1884–1951), and Jakub Parnas (1884–1949).
Glycolysis using the EMP pathway consists of two distinct phases. The first part of the pathway,
called the energy investment phase, uses energy from two ATP molecules to modify a glucose
molecule so that the six-carbon sugar molecule can be split evenly into two phosphorylated
three-carbon molecules called glyceraldehyde 3-phosphate (G3P). The second part of the
pathway, called the energy payoff phase, extracts energy by oxidizing G3P to pyruvate,
producing four ATP molecules and reducing two molecules of NAD+ to two molecules of
NADH, using electrons that originated from glucose.

However, some prokaryotes use alternative glycolytic pathways.

Entner-Doudoroff (ED) pathway, named after its discoverers Nathan Entner and Michael
Doudoroff (1911–1975).
Although some bacteria, including the opportunistic gram-negative pathogen Pseudomonas
aeruginosa, contain only the ED pathway for glycolysis, other bacteria, like E. coli, have the
ability to use either the ED pathway or the EMP pathway.
Pentose phosphate pathway (PPP) also called the phosphogluconate pathway or the hexose
monophosphate shunt. Evidence suggests that the PPP may be the most ancient universal
glycolytic pathway. The intermediates from the PPP are used for the biosynthesis of nucleotides
and amino acids. Therefore, this glycolytic pathway may be favored when the cell has need for
nucleic acid and/or protein synthesis, respectively.
Transition Reaction, Coenzyme A, and the Krebs Cycle
Glycolysis produces pyruvate, which can be further oxidized to capture more energy. For
pyruvate to enter the next oxidative pathway, it must first be decarboxylated by the enzyme
complex pyruvate dehydrogenase to a two-carbon acetyl group in the transition reaction, also
called the bridge reaction 
In the transition reaction, electrons are also transferred to NAD+ to form NADH. To proceed to
the next phase of this metabolic process, the comparatively tiny two-carbon acetyl must be
attached to a very large carrier compound called coenzyme A (CoA). The transition reaction
occurs in the mitochondrial matrix of eukaryotes; in prokaryotes, it occurs in the cytoplasm
because prokaryotes lack membrane-enclosed organelles.
he Krebs cycle transfers remaining electrons from the acetyl group produced during the
transition reaction to electron carrier molecules, thus reducing them. The Krebs cycle also occurs
in the cytoplasm of prokaryotes along with glycolysis and the transition reaction, but it takes
place in the mitochondrial matrix of eukaryotic cells where the transition reaction also occurs.
The Krebs cycle is named after its discoverer, British scientist Hans Adolf Krebs (1900–1981)
and is also called the citric acid cycle, or the tricarboxylic acid cycle (TCA) because citric acid
has three carboxyl groups in its structure. Unlike glycolysis, the Krebs cycle is a closed loop:
The last part of the pathway regenerates the compound used in the first step (Figure 4). The eight
steps of the cycle are a series of chemical reactions that capture the two-carbon acetyl group (the
CoA carrier does not enter the Krebs cycle) from the transition reaction, which is added to a
four-carbon intermediate in the Krebs cycle, producing the six-carbon intermediate citric acid
(giving the alternate name for this cycle). As one turn of the cycle returns to the starting point of
the four-carbon intermediate, the cycle produces two CO 2 molecules, one ATP molecule (or an
equivalent, such as guanosine triphosphate [GTP]) produced by substrate-level phosphorylation,
and three molecules of NADH and one of FADH2.

Although many organisms use the Krebs cycle as described as part of glucose metabolism,
several of the intermediate compounds in the Krebs cycle can be used in synthesizing a wide
variety of important cellular molecules, including amino acids, chlorophylls, fatty acids, and
nucleotides; therefore, the cycle is both anabolic and catabolic (Figure 5).
Cellular Respiration
We have just discussed two pathways in glucose catabolism—glycolysis and the Krebs cycle—
that generate ATP by substrate-level phosphorylation. Most ATP, however, is generated during a
separate process called oxidative phosphorylation, which occurs during cellular respiration.
Cellular respiration begins when electrons are transferred from NADH and FADH 2—made in
glycolysis, the transition reaction, and the Krebs cycle—through a series of chemical reactions to
a final inorganic electron acceptor (either oxygen in aerobic respiration or non-oxygen inorganic
molecules in anaerobic respiration). These electron transfers take place on the inner part of the
cell membrane of prokaryotic cells or in specialized protein complexes in the inner membrane of
the mitochondria of eukaryotic cells. The energy of the electrons is harvested to generate an
electrochemical gradient across the membrane, which is used to make ATP by oxidative
phosphorylation.
Electron Transport System
The electron transport system (ETS) is the last component involved in the process of cellular
respiration; it comprises a series of membrane-associated protein complexes and associated
mobile accessory electron carriers. Electron transport is a series of chemical reactions that
resembles a bucket brigade in that electrons from NADH and FADH 2 are passed rapidly from
one ETS electron carrier to the next. These carriers can pass electrons along in the ETS because
of their redox potential. For a protein or chemical to accept electrons, it must have a more
positive redox potential than the electron donor. Therefore, electrons move from electron carriers
with more negative redox potential to those with more positive redox potential. The four major
classes of electron carriers involved in both eukaryotic and prokaryotic electron transport
systems are the cytochromes, flavoproteins, iron-sulfur proteins, and the quinones.
In aerobic respiration, the final electron acceptor (i.e., the one having the most positive redox
potential) at the end of the ETS is an oxygen molecule (O 2) that becomes reduced to water (H2O)
by the final ETS carrier. This electron carrier, cytochrome oxidase, differs between bacterial
types and can be used to differentiate closely related bacteria for diagnoses. For example, the
gram-negative opportunist Pseudomonas aeruginosa and the gram-negative cholera-
causing Vibrio cholerae use cytochrome c oxidase, which can be detected by the oxidase test,
whereas other gram-negative Enterobacteriaceae, like E. coli, are negative for this test because
they produce different cytochrome oxidase types.
There are many circumstances under which aerobic respiration is not possible, including any one
or more of the following:
 The cell lacks genes encoding an appropriate cytochrome oxidase for transferring electrons
to oxygen at the end of the electron transport system.
 The cell lacks genes encoding enzymes to minimize the severely damaging effects of
dangerous oxygen radicals produced during aerobic respiration, such as hydrogen peroxide
(H2O2) or superoxide (O−2)(O2−).
 The cell lacks a sufficient amount of oxygen to carry out aerobic respiration.
One possible alternative to aerobic respiration is anaerobic respiration, using an inorganic
molecule other than oxygen as a final electron acceptor. There are many types of anaerobic
respiration found in bacteria and archaea. Denitrifiers are important soil bacteria that use
nitrate (NO−3)(NO3−) and nitrite (NO−2)(NO2−) as final electron acceptors, producing nitrogen
gas (N2). Many aerobically respiring bacteria, including E. coli, switch to using nitrate as a final
electron acceptor and producing nitrite when oxygen levels have been depleted.
Microbes using anaerobic respiration commonly have an intact Krebs cycle, so these organisms
can access the energy of the NADH and FADH 2 molecules formed. However, anaerobic respirers
use altered ETS carriers encoded by their genomes, including distinct complexes for electron
transfer to their final electron acceptors. Smaller electrochemical gradients are generated from
these electron transfer systems, so less ATP is formed through anaerobic respiration.
Chemiosmosis, Proton Motive Force, and Oxidative Phosphorylation
In each transfer of an electron through the ETS, the electron loses energy, but with some
transfers, the energy is stored as potential energy by using it to pump hydrogen ions (H+) across a
membrane. In prokaryotic cells, H+ is pumped to the outside of the cytoplasmic membrane
(called the periplasmic space in gram-negative and gram-positive bacteria), and in eukaryotic
cells, they are pumped from the mitochondrial matrix across the inner mitochondrial membrane
into the intermembrane space. There is an uneven distribution of H + across the membrane that
establishes an electrochemical gradient because H+ ions are positively charged (electrical) and
there is a higher concentration (chemical) on one side of the membrane. This electrochemical
gradient formed by the accumulation of H+ (also known as a proton) on one side of the
membrane compared with the other is referred to as the proton motive force (PMF). Because
the ions involved are H+, a pH gradient is also established, with the side of the membrane having
the higher concentration of H+ being more acidic. Beyond the use of the PMF to make ATP, as
discussed in this chapter, the PMF can also be used to drive other energetically unfavorable
processes, including nutrient transport and flagella rotation for motility.
The potential energy of this electrochemical gradient generated by the ETS causes the H + to
diffuse across a membrane (the plasma membrane in prokaryotic cells and the inner membrane in
mitochondria in eukaryotic cells). This flow of hydrogen ions across the membrane,
called chemiosmosis, must occur through a channel in the membrane via a membrane-bound
enzyme complex called ATP synthase (Figure 1). The tendency for movement in this way is
much like water accumulated on one side of a dam, moving through the dam when opened. ATP
synthase (like a combination of the intake and generator of a hydroelectric dam) is a complex
protein that acts as a tiny generator, turning by the force of the H + diffusing through the enzyme,
down their electrochemical gradient from where there are many mutually repelling H + to where
there are fewer H+. In prokaryotic cells, H+ flows from the outside of the cytoplasmic membrane
into the cytoplasm, whereas in eukaryotic mitochondria, H + flows from the intermembrane space
to the mitochondrial matrix. The turning of the parts of this molecular machine regenerates ATP
from ADP and inorganic phosphate (Pi) by oxidative phosphorylation, a second mechanism for
making ATP that harvests the potential energy stored within an electrochemical gradient.
The number of ATP molecules generated from the catabolism of glucose varies. For example,
the number of hydrogen ions that the electron transport system complexes can pump through the
membrane varies between different species of organisms. In aerobic respiration in mitochondria,
the passage of electrons from one molecule of NADH generates enough proton motive force to
make three ATP molecules by oxidative phosphorylation, whereas the passage of electrons from
one molecule of FADH2 generates enough proton motive force to make only two ATP
molecules. Thus, the 10 NADH molecules made per glucose during glycolysis, the transition
reaction, and the Krebs cycle carry enough energy to make 30 ATP molecules, whereas the two
FADH2 molecules made per glucose during these processes provide enough energy to make four
ATP molecules. Overall, the theoretical maximum yield of ATP made during the complete
aerobic respiration of glucose is 38 molecules, with four being made by substrate-level
phosphorylation and 34 being made by oxidative phosphorylation (Table 1). In reality, the total
ATP yield is usually less, ranging from one to 34 ATP molecules, depending on whether the cell
is using aerobic respiration or anaerobic respiration; in eukaryotic cells, some energy is expended
to transport intermediates from the cytoplasm into the mitochondria, affecting ATP yield.