PHARMACEUTICAL BIOCHEMISTRY (LEC)

ENZYMES & INTRODUCTION TO NUCLEIC ACIDS

ENZYMES Vitamins as Coenzymes

• Proteins that increase the rate of chemical Vitamin Coenzyme Chemical Groups
reactions without being changed in the overall Transferred
process Thiamine thiamine Aldehydes
(B1) pyrophosphate
• Essential for breakdown of nutrients that are used
(TPP)
to supply energy and chemical building blocks
Riboflavin flavin adenine Electrons
• Catalytic activity depends on their native protein (B2) dinucleotide (FAD)
conformation Niacin nicotinamide Hydride ion (:H-)
(B3) adenine
ENZYMES are biological catalysts
dinucleotide (NAD+)
• The basic function of an enzyme is to increase the Pantothenic acid coenzyme A (CoA) Acyl groups
rate of a reaction (B5)
• Most enzymes act specifically with only one Pyridoxine (B6) Pyridoxal phosphate Amino groups
reactant (called a substrate) to produce products) Folic Acid (B9) Tetrahydrofolate One-carbon
groups
Properties and Terminologies Cyanocobalamin Coenzyme B-12 (5’- H atoms and
(B12) deoxyadenosylcobal alkyl groups
▪ Most enzymes are proteins amin)
▪ Some enzymes require no chemical groups for Biotin (H) Biocytin CO2
activity other than their amino acid residues
▪ However – Enzymes may require a non-peptide
component for their activity HOLOENZYME
o Cofactor – either one or more inorganic
• Catalytically active enzyme together with its
ions
bound coenzyme and/or metal ions
o Coenzyme – complex organic or
❖ Protein Part – APOENZYME (aka
metalloorganic molecule
APOPROTEIN)
COFACTORS ❖ Inactive form – PROENZYME (aka
Zymogen)
Metal
Zn2+ Carbonic anhydrase
Zn2+ Carboxypeptidase
Mg2+ EcoRV
Mg2+ Hexokinase
Ni2+ Urease
Mo Nitrate reductase NOMENCLATURE
Se Glutathione peroxidase
• The commonly used names for most enzymes
Mn2+ Superoxide dismutase
describe the type of reaction catalyzed, followed
K+ Propionyl CoA carboxylase
by the suffix -ase.
▪ Dehydrogenases – remove hydrogen
On COENZYMES: atoms (oxidation)
▪ Proteases – hydrolyze proteins
▪ Cosubstrate - acts as transient carriers of specific ▪ Isomerases – catalyze rearrangement in
functional groups configuration
➢ Derived from vitamins, organic nutrients
• Modifiers may precede the name to indicate:
▪ Prosthetic group – if a coenzyme or a cofactor is
• The substrate (xanthine oxidase)
very tightly or even covalently bound to the
• The source of the enzyme (pancreatic
enzyme protein
ribonuclease)
• Its regulation (hormone-sensitive lipase)
• A feature of its mechanism of action
(cysteine protease)
 • Alphanumeric designators may be added to
identify multiple forms of an enzyme
o The polymerase is essential for the
➢ RNA polymerase ///; protein kinase Cβ
synthesis of DNA and RNA
• Some enzymes retain their original trivial names,
which give no hint of the associated enzymatic
reaction
➢ Pepsin, trypsin, and chymotrypsin which
catalyze the hydrolysis of proteins

NOMENCLATURE: INTERNATIONAL AGREEMENT

• A system adopted in naming and classifying

enzymes
• Enzymes are classified into 6 classes based on the
International Union of Biochemistry and Molecular
Biology (IUBMB):
Six major classes of enzymes OTHLIL

Class Type of reaction Example

1. Oxidoreductases Oxidation-reduction Lactate
dehydrogenase
2. Transferases Group transfer Nucleoside
monophosphate
kinase (NMP
kinase)
3. Hydrolases Hydrolysis reactions Chymotrypsin 3. HYDROLASES – amide, ester FG
(transfer of functional o Catalyze hydrolysis reactions where a
groups to water) molecule is split into two or more smaller
4. Lyases Addition or removal Fumarase molecules by the addition of water
(separation) of groups to form ▪ PROTEASES split protein
double bonds molecules
5. Isomerases Isomerization Triose phosphate HIV protease is essential
(intramolecular group isomerase for HIV replication
transfer) Caspase plays a major
6. Ligases (merging) Ligation of two Aminoacyl-tRNA role in apoptosis
substrates at the synthetase
▪ NUCLEASES split nucleic acids
expense of ATP
(DNA and RNA) – break
hydrolysis
phosphodiester bond
Based on the substrate
• Example of formal systematic name: type, they are divided
into RNase and DNase.
2.7.1.1 o RNase
(2) – Transferase catalyzes the
hydrolysis of
Classification of Enzymes RNA
o DNase acts on
1. OXIDOREDUCTASES
DNA
o Catalyze a variety of oxidation-reduction
reactions NUCLEASES – PDE (nucleotides)
o Common names include dehydrogenase,
oxidase, reductase and catalase ❖ They may also be divided into
2. TRANSFERASES exonuclease and endonuclease
o Catalyze transfers of groups (acetyl, - The exonuclease progressively
methyl, phosphate (kinase), etc.) splits off single nucleotides from
o Common names include one end of DNA or RNA
acetyltransferase, methylase, protein - The endonuclease splits DNA or
kinase and polymerase (DNA & RNA; RNA at internal sites
transfer of nitrogenous bases) ❖ PHOSPHATASE catalyzes
o The first three subclasses play major roles dephosphorylation (removal of phosphate
in the regulation of cellular processes groups) kinase
 - Example: calcineurin
- The immunosuppressive drugs
FK506 and Cyclosporin A are the
inhibitors of calcineurin
4. LYASES
o Catalyze the cleavage of C-C, C-O, C-S and
C-N bonds by means other than hydrolysis
or oxidation
o Common names include decarboxylase
and aldolase
5. ISOMERASES
o Catalyze atomic rearrangements within a
molecule
o Examples include rotamase, protein
disulfide isomerase (PDI), epimerase and
racemase
6. LIGASES
o Catalyze the reaction which joins two
molecules
o Examples include peptide synthase,
aminoacyl-tRNA synthetase, DNA ligase
and RNA ligase

Mechanism of Enzyme Action

▪ Enzyme-catalyzed reaction takes place within a

pocket on the enzyme – ACTIVE SITE
▪ SUBSTRATE – the molecule that is bound to the
active site and acted upon by the enzyme

Catalytic Power of Enzymes

▪ Catalytic Power
o Rate enhancements that enzymes bring
about are in the range of 5-17 orders of
magnitude
▪ HOW and WHY? What is the source of the energy
for lowering the activation energy?

1. Rearrangement of covalent bonds during an

2.
▪ The function of enzymes is to increase the RATE of
a reaction, not equilibria
▪ Reaction equilibria – means that there is no net
change in the concentrations of the reactants and
products
enzyme-catalyzed reaction
- Formation of transient covalent bond
with a substrate
- Transient transferring of chemical group
from the substrate to the enzyme
- Usually happens in the active site
Non-covalent interactions between enzyme and
substrate
- Much of the energy required to lower
activation energies is derived from weak,
noncovalent interactions between the
enzyme and substrate
- Mediated by the same forces that
stabilize protein structure

Binding Energy

▪ The energy derived from enzyme-substrate

interaction
 ▪ Energy released that stabilizes the interaction
▪ Major source of free energy used by enzymes to
lower the activation energies of reactions
▪ Contributes to specificity as well as to catalysis

Specificity of Enzymes

Specificity

• Vey specific; readily discriminating between

substrates with quite similar structures
o Induced Fit Theory
o Lock and Key Hypothesis

Induced Fit Theory

• Postulated by Daniel Koshland

• It states that, when substrates
approach and bind to an enzyme
they induce a conformational
change
• This change is analogous to
placing a hand (substrate) into a
glove (enzyme)

Lock and Key Theory

• First postulated in 1894

by Emil Fischer
• The lock is the enzyme
Key=substrate and the key is the substrate
Lock=enzyme
• Only the correctly sized MECHANISMS TO FACILITATE CATALYSIS
Correct key (substrate) fits into the
A. ACID-BASE CATALYSIS
fit, key hole (active site) of the
Will react lock (enzyme) o Refers to proton transfers mediated by other
classes of molecules
o General or Specific
o The active sites of some enzymes contain amino
Incorrect No reaction acid functional groups that can participate in
substrate
catalysis as proton donors or acceptors
Lock and Key Hypothesis B. COVALENT CATALYSIS
▪ Enzymes were structurally complementary to their o Involves the formation of a transient covalent
substrates – perfect fit bond between the enzyme and one or more
▪ May be misleading when applied to enzyme substrates
catalysis o Common among enzymes that catalyze group
▪ “an enzyme completely complementary to its transfer reactions
substrate would be a very poor enzyme”
C. METAL ION CATALYSIS
Instead:
o Ionic interactions between an enzyme-bound
o An enzyme must be complementary to the metal and a substrate can help orient the
reaction transition state substrate for reaction or stabilize charged reaction
o Meaning, optimal interactions (weak states
interactions) between the enzyme and o Metals can also mediate oxidation-reduction
substrate occur only in the transition state reaction by reversible changes in the metal ion’s
oxidation state
Chymotrypsin Michaelis-Menten Kinetics

• Combination of covalent catalysis, general acid- • States that enzyme first combines reversibly with
base catalysis, and transition-state stabilization its substrate to form an enzyme-substrate complex
in a relatively FAST reversible step:
FACTORS AFFECTING ENZYME REACTIONS

I. SUBSTRATE CONCENTRATION

• The rate of enzyme catalyzed

reaction increases with substrate
concentration until a maximal • The ES complex then breaks down in a SLOWER
velocity (Vmax) is reached second step to yield the free enzyme and the
reaction product P:

Effect of temperature

▪ Higher temperature increases the rate of

a reaction until a peak velocity
▪ Above a certain temperature, activity
begins to decline because the enzyme
begins to denature
▪ The rate of chemical reactions therefore
increases with temperature but then
decreases
Effect of pH
▪ Each enzyme has an optional pH
▪ In order to ineract, the E and S
have specific chemical groups in
ionized or unionized state
▪ Amino group in protonated form
(-NH3+) → increase catalytic
activity
▪ At alkaline pH, amino group is
deprotonated → decrease in rate of reaction
▪ Extremes of pH can lead to denaturation
Assumptions
• Relative concentrations of E and S
➢ [S] > [E], so [ES] at any time is small
• Steady-state assumption
➢ Pre-steady state: [ES] builds up (transient)
➢ Steady State: [ES] does not change in time
E + S = ES = E + P
• Initial velocity
➢ Used in the analysis of enzyme reactions
➢ Rate of reaction is measured as soon as E
and S are mixed
➢ [P] is very small, the rate of back reaction
from P to S can be ignored
Michaelis-Menten Kinetics

ENZYME KINETICS

The field of biochemistry concerned with the quantitative

measurement of the rates of enzyme-catalyzed reactions
and the systematic study of factors that affect these rates.

Plot Curve: Enzyme Kinetics

Velocity of a reaction (V)

– defined as the number
of substrate molecules
converted to product per
unit time (µmol of product
formed/unit time)
Michaelis-Menten Kinetics

• Vmax is observed when virtually all the enzyme is

present as the ES complex and [E] is vanishingly
small = SATURATED
• Saturation exists when [S] is sufficiently high that
essentially all the free enzyme has been converted
to the ES form.
➢ Responsible for the plateau observed in
the figure earlier.

CONCLUSIONS

1. Characteristics of Km
INHIBITION OF ENZYME ACTIVITY
a. Small Km – reflects high affinity of the E for S
because a low concentration of S Is needed to half- INHIBITOR → substance that can diminish the velocity of an
saturate the enzyme – that is, reach a velocity that enzyme catalyzed reaction
is ½ Vmax
b. Large Km – reflects low affinity of E for S because a
high concentration of S is needed to half-saturate
the enzyme

2. Relationship of velocity to enzyme concentration

• The rate of reaction is directly proportional to the

enzyme concentration at all substrate
concentrations

3. Order of reaction

• First order – [S] < Km, the velocity of reaction is Inhibition of Enzyme Activity
roughly proportional to the substrate
concentration TYPES OF REVERSIBLE INHIBITION
• Zero order – [S] > Km, the velocity is constant and
1. COMPETITIVE INHIBITION
equal to Vmax; the rate of reaction is then
independent of substrate concentration 2. UNCOMPETITIVE INHIBITION

3. NONCOMPETITIVE INHIBITION

Competitive Inhibition

o A competitive inhibitor competes with the

substrate for the active site of an enzyme
o Many competitive inhibitors are structurally
similar to the substrate and combine with the
enzyme to form an EI complex, but without leading
to catalysis

Example:

▪ Statins – competitively inhibit HMG-CoA

reductase
o Structural analogs of the natural
substrate for the enzyme and
compete effectively to inhibit
HMG CoA reductase
Uncompetitive Inhibition
o An uncompetitive inhibitor binds at the site
distinct from the substrate active site
o Binds only to the ES complex
o Example:
Lansoprazole – inhibits alkaline phosphatase
Noncompetitive Inhibition
o Rarely encountered in experiments
o A mixed inhibitor also binds at a site distinct from
the substrate active site, but it binds to either E or
ES.
o Example:
Oxypurinol – metabolite of allopurinol
INDUCTION and
REPRESSION of Enzyme Synthesis
• Alter the total population of active sites rather
than influencing the efficiency of existing enzyme
molecules
• Enzymes that are needed at only one stage of
development or under selected physiologic
conditions are subject to regulation of enzyme
synthesis
• Enzymes that are in constant use are not regulated
by altering the rate of enzyme synthesis
ENZYMES IN CLINICAL USE
Enzymes inhibitors as DRUGS
In CLINICAL DIAGNOSIS
Enzyme inhibitors as DRUGS
• STATINS – HMG (3-hydroxy-3-methylglutaryl)
Coenzyme A reductase inhibitors; lower serum
lipid concentration
• EMTRICTABINE and TENOFOVIR DISOPROXIL
FUMARATE – inhibitors of viral reverse
transcriptase; block replication of HIV
• ACE Inhibitors (Captopril, Lisinopril, Enalapril) –
antihypertensive agents
• Lactam Antibiotics (penicillin and Amoxicillin) –
inhibitors of alanyl-alanine carboxypeptidase-
transpeptidase, thus blocking cell wall synthesis
 Principal Serum Enzymes Used in Clinical Diagnosis

Serum Enzyme Major Diagnostic Use

Aminotransferases
▪ Aspartate Myocardial infaction
aminotransferase
(AST, or SGOT)

▪ Alanine Viral hepatitis

aminotransferase
(ALT, or SGPT)
Amylase Acute pancreatitis
Ceruplasmin Hepatolenticular degeneration
(Wilson’s disease)
Creatine kinase Muscle disorders and myocardial
infection
y-Glutamyl transferase Various liver diseases
Lactate dehydrogenase Liver disease
(isoenzymes-5)

Lactate dehydrogenase (isozyme – Myocardial infaction

1)
Lipase Acute pancreatitis
Phosphatase, acid Metastatic carcinoma of the
prostate

Phosphatase, alkaline Various bone disorders,

obstructive liver diseases