Sometimes the enzyme alone isn’t enough to get a reaction going.

Cofactors
Cofactors are small molecules that an enzyme needs in order to work.
Such enzymes are called apoenzymes without their cofactors, but complete,(aponzymes
are the protein portion of an enzyme once they are bound to a cofactor they become
active. catalytically active enzymes are called holoenzymes.)
Cofactors can either be organic or inorganic, with inorganic metal ions being the most
common. Cofactors bind to and are released from an enzyme’s active site, just like a
substrate.
Cofactors can be divided into two major groups:
organic Cofactors, such as flavin or heme,
inorganic cofactors, such as the metal ions Mg2+, Cu+, Mn2+, or iron-sulfur clusters.
 (In organic) Examples of enzymes containing this ion
Ion

Cupric Cytochrome oxidase

Catalase
Ferrous or Ferric Cytochrome (via Heme)
Nitrogenase
Hydrogenase

Glucose 6-phosphatase
Magnesium Hexokinase
DNA polymerase

Manganese Arginase

Nitrate reductase
Molybdenum Nitrogenase
Xanthine oxidase

Nickel Urease

Alcohol dehydrogenase
Zinc Carbonic anhydrase
DNA polymerase
Cofactor Vitamin Additional component Chemical group(s) transferred Distribution

Thiamine (B1) Bacteria, archaea and

Thiamine pyrophosphate[31] pyrophosphate 2-carbon groups, α cleavage
eukaryotes

Niacin (B3) Bacteria, archaea and

NAD+ and NADP+[32] ADP Electrons
eukaryotes

Pyridoxine (B6) Bacteria, archaea and

Pyridoxal phosphate[33] None Amino and carboxyl groups
eukaryotes

Vitamin B12 Bacteria, archaea and

Methylcobalamin[34] Methyl group acyl groups
eukaryotes

Cobalamine (B12) Bacteria, archaea and

Cobalamine[5] None hydrogen, alkyl groups
eukaryotes

CO2 Bacteria, archaea and

Biotin[35] Biotin (H) None
eukaryotes

Pantothenic acid (B5) Acetyl group and other Bacteria, archaea and
Coenzyme A[36] ADP
acyl groups eukaryotes

Folic acid (B9) Methyl, formyl, methylene and Bacteria, archaea and
Tetrahydrofolic acid[37] Glutamate residues
formimino groups eukaryotes
Bacteria, archaea and
Menaquinone[38] Vitamin K None Carbonyl group and electrons
eukaryotes
Bacteria, archaea and
Ascorbic acid[39] Vitamin C None Electrons
eukaryotes

Riboflavin (B2) Bacteria, archaea and

Flavin mononucleotide[40] None Electrons
eukaryotes

Riboflavin (B2) Bacteria, archaea and

Flavin adenine dinucleotide[40] ADP Electrons
eukaryotes

Riboflavin (B2) Methanogens and some

Coenzyme F420[41] Amino acids Electrons
bacteria
coenzymes
• Coenzyme is any of a diverse group of small organic, non-protein, freely diffusing
molecules that are loosely associated with and essential for the activity of
enzymes, serving as carrier molecules that transfer chemical groups. Coenzymes
are sometimes referred to as cosubstrates. These molecules are substrates for
enzymes and do not form a permanent part of the enzymes' structures.
• The term coenzymes is sometimes defined in such a way as to include prosthetic
groups (Alberts et al. 1989; Bender and Bender 2005; McGraw-Hill 2005).
• However, prosthetic groups are non-protein components that are bound
tightly (covalently linked) to enzymes—such as iron-sulfur centers, flavin, or haem
groups.
Vitamin coenzyme
Additional Chemical group(s)
Coenzyme Vitamin component transferred Distribution

NAD+ and NADP+ [5] Niacin (B3) ADP Electrons Bacteria, archaea
and eukaryotes

Pantothenic acid (B5) Acetyl group and Bacteria, archaea

Coenzyme A [9] ADP other acyl groups and eukaryotes

Methyl, formyl,
Tetrahydrofolic acid Folic acid (B9) Glutamate residues methylene and Bacteria, archaea
[10]
formimino groups and eukaryotes

Carbonyl group and Bacteria, archaea

Menaquinone [11] Vitamin K None electrons and eukaryotes
Bacteria, archaea
Ascorbic acid [12] Vitamin C None Electrons and eukaryotes

Coenzyme F420 [13] Riboflavin (B2) Amino acids Electrons Methanogens and
some bacteria
Non vitamin

Coenzyme Chemical group(s) transferred Distribution

Adenosine triphosphate [14] Phosphate group Bacteria, archaea and eukaryotes
S-Adenosyl methionine [15] Methyl group Bacteria, archaea and eukaryotes

3'-Phosphoadenosine-5'-phosphosulfate [16] Sulfate group Bacteria, archaea and eukaryotes

Coenzyme Q [17] Electrons Bacteria, archaea and eukaryotes

Tetrahydrobiopterin [18] Oxygen atom and electrons Bacteria, archaea and eukaryotes

Cytidine triphosphate [19] Diacylglycerols and lipid head groups Bacteria, archaea and eukaryotes

Nucleotide sugars [20] Monosaccharides Bacteria, archaea and eukaryotes

Glutathione [21][22] Electrons Some bacteria and most eukaryotes

Coenzyme M [23][24] Methyl group Methanogens

Coenzyme B [25] Electrons Methanogens
Methanofuran [26] Formyl group Methanogens
Tetrahydromethanopterin [27] Methyl group Methanogens
• The International Union of Pure and Applied Chemistry (IUPAC) draws
a distinction between coenzymes and prosthetic groups. IUPAC
defines a coenzyme as a low-molecular-weight, non-protein organic
compound that is loosely attached, participating in enzymatic
reactions as a dissociable acceptor of chemical groups or electrons;
• a prosthetic group is defined as a tightly bound, nonpolypeptide unit
in a protein (IUPAC 1997a, 1997b). Both coenzymes and prosthetic
groups are types of the broader group of cofactors, which are any
non-protein molecules (usually organic molecules or metal ions) that
are required by an enzyme for its activity (IUPAC 1997c).
Role of coenzyme
• The function of coenzymes is to transport groups between enzymes.
• Chemical groups include hydride ions which are carried by coenzymes
such as NAD,
• phosphate groups which are carried by coenzymes such as ATP
• acetyl groups which are carried by coenzymes such as coenzyme A.
• Coenzymes which lose or gain these chemical groups in the course of
the reaction are often reformed in the same metabolic pathway. For
example NAD+ used in glycolysis and the citric acid cycle is replaced in
the electron transport chain
Function of coenzyme
• The coenzyme is essential for the biological activity of the enzyme.
• A coenzyme is a low molecular weight organic substance, without
which the enzyme cannot exhibit any reaction.
• One molecule of the coenzyme is able to convert a large number of
substrate molecules with the help of enzyme.
Types
• Vitamins
NAD
NADP
coenzyme a
ascorbic acid
vit k
riboflavin b12

• Non vitamin
• nucleotides, such as adenosine triphosphate, S-Adenosyl methionine
• 3'-Phosphoadenosine-5'-phosphosulfate Coenzyme Q
• Cytidine triphosphate
prosthetic group
• prosthetic group is a tightly bound, specific non-polypeptide unit
required for the biological function of some proteins. The prosthetic
group is inorganic (such as a metal ion (iron) ), but is not composed of
amino acids. Prosthetic groups are bound tightly to proteins and may
even be attached through a covalent bond,
General characteristic of enzymatic reaction
[Link] efficiency
Enzymes are highly efficient. They convert the substrate into product in
minutes.
2. Specificity
enzymes have a specific shape, therefore only a certain substrate will fit
its active site.
There are two theories of enzyme action: Lock and Key and Induced
Fit.
Lock-and-Key model 1894 by Emil Fischer

- Enzyme has a rigid, nonflexible shape.

- An enzyme binds only substrates that

exactly fit the active site.

-The enzyme is analogous to a lock.

- The substrate is the key that fits into the lock

Induced-Fit model Daniel Koshland's 1958

1. Why Enzyme-Substrate Complex is not stable?

(no reason for the reaction to occur)

Problems:
2. X-ray diffraction: size and shape of the actice site
chanegs when a substrate enters.
 Induced-Fit model
- Enzyme structure is flexible, not rigid.

- Enzyme and substrate adjust the shape

of the active site to bind substrate.

- The range of substrate specificity

increases.
- A different substrate could not induce
these structural changes and no
catalysis would occur.
- For example, the binding of glucose to
hexokinase induces a conformational
change in the structure of the enzyme
such that the active site assumes a shape
that is complementary to the substrate
(glucose) only after it has bound to the
enzyme.
 Factors affecting enzyme activity

Activity of enzyme: how fast an enzyme catalyzes the reaction.

Effect of temp
pH
time
substrate concentration
Enzyme concentration
Enzyme activator / inhibition
 Temperature

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

- At low T, enzyme shows little activity (not an enough amount of energy for
the catalyzed reaction).

- At very high T, enzyme is destroyed (tertiary structure is denatured).

- Optimum temperature: 35°C or body temperature.

- Storage of enzymes at 5·C or below is generally the most suitable.
- Some enzymes lose their activity when frozen
pH
• Enzymes are affected by changes in pH.
• Enzymes according to ph divided into three types. acidic, basic and neutrophilic
.
• The most favorable pH value - the point where the enzyme is most active - is
known as the optimum pH.
• Extremely high or low pH values generally result in complete loss of activity for
most enzymes.
• pH is also a factor in the stability of enzymes. As with activity, for each enzyme
there is also a region of pH optimal stability.
• Digestive enzyme(pepsin trypsin chymotrypsin ) activate at acid ph
• Food industry enzyme activate at neutral pH
• Chemical industry enzyme activate at alkaline ph.
 Time

• The rate of enzyme reaction is directly proportional to

the time period but after optimum time period the rate
of reaction decreases. This decreases in enzyme
reaction may be suggested to the denaturation of
enzyme or presence of natural inhibition.
 Enzyme concentration
• The rate of enzymatic reaction increase with the
increase of enzyme concentration upto certain limits
and than remain constant (because the extra enzyme
is not capable to react with substrate)
• The rate of reaction increase upto certain limits and
then decrease with increase of enzyme
concentration(due natural inhibitor that occupy the
enzyme active center and decrease the interaction of
substrate and enzyme
 Substrate concentration
• In the presence of a given amount of enzyme, the rate of
enzymatic reaction increases as the substrate concentration
increases until a limiting rate is reached, after which further
increase in the substrate concentration produces no
significant change in the reaction rate. At this point, so
much substrate is present that essentially all of the enzyme
active sites have substrate bound to them.
• In other words, the enzyme molecules are saturated with
substrate. The excess substrate molecules cannot react until
the substrate already bound to the enzymes has reacted and
been released (or been released without reacting).
 Inhibitors/activators
• Inhibitors are chemicals that reduce the rate of enzymatic
reactions.
• The are usually specific and they work at low concentrations.
• They block the enzyme but they do not usually destroy it.
• Since blocking an enzyme's activity can kill a pathogen or correct a
metabolic imbalance, many drugs are enzyme inhibitors.
 Types of inhibitors

Irreversible inhibitors Reversible inhibitors

Competitive Noncompetitive Uncompetitive

 Irreversible inhibitors Reversible inhibitors

Type of bonds Inhibitors bind covalently Inhibitors bind non-

with E with enzyme covalently with enzyme

Removal Cannot be removed by Can be removed by

dialysis or other way dialysis

Activity Permanently modify the Removal of the inhibitor

Restoration active site residues(functional restores enzyme activity
group) which the enzyme
become inactive.
It is relatively simple to distinguish the three types of reversible inhibition by
comparing the Michaelis-Menten and Lineweaver-Burke kinetics (Vmax and
Km) in the presence and absence of the inhibitor.
Vmax . The rate of reaction when the enzyme is saturated with substrate is the
maximum rate of reaction,
Km is the concentration of substrate which permits the enzyme to achieve
half Vmax
Competitive inhibitors
• As the name implies, the inhibitor compete with the
substrate for active site of the enzyme.

• The substrate and inhibitors have resemble structures

• Have the same Vmax (with I OR without I)

• Km high and affinity low (with I)

• This type of inhibition can be overcome by sufficiently

high concentrations of substrate by out-competing
the inhibitor
NONCompetitive inhibitors

• The inhibitors bind with site on the enzyme

other than active site

• They can bind with E or ES complex.

• Have the same Km (with I OR without I)

• low Vmax (with I)

Cynide and hydrogen sulphide ..strongly inhibit the action of iron containing
enzyme like catalase and peroxidase
 UNCompetitive inhibitorsMPETITIVE
INHIBITORS
• The inhibitor binds only to the substrate-
enzyme complex
• Both Vmax and Km are low (with I)

Example
hydrazine inhibit aryl sulphate
• Phenylalanine inhibit alkaline phosphatase
 Irreversible inhibitor
• Iodoacetamide …irreversibly inhibit the catalytic activity of
some enzyme by modifyfing cysteine and other side chain
• Diisopropyl-phosphoflouridate (DIPF) is protein
irreversible inhibitor of enzyme that active scryl residue at
their catalytic side
Importance of Enzyme Inhibition

• For understanding the regulation of enzyme activity within the living cells

• Useful in elucidating the cellular metabolic pathways by causing accumulation of intermediates

• Identification of the catalytic / functional groups at the active site of E

• Provide information about substrate specificity of the enzyme

• Useful to study the mechanism of catalytic activity

• Enzyme inhibitors have therapeutic applications – some drugs useful in medicine appear to function by
inhibiting certain E.
• Most drugs are Competitive or Suicide inhibitors.