Enzymes

Enzymes
Ashikh Seethy
Dept of Biochemistry
Maulana Azad Medical College
New Delhi

Enzymes are Biological Catalysts
• Selectively and efficiently catalyze biological
reactions
• Most of them are proteins
• Very specific
• Works within narrow ranges of pH and
temperature
• Abnormality can lead to diseases
• Used in diagnosis, treatment and prognosis of
diseases
• Chemical engineering, agriculture, food
technology, research

History of
Enzymes:
• Late 1700s and early 1800s:
 Digestion of meat by stomach secretions
 Conversion of starch to sugars by plant extracts
and saliva
• 1850: Louis Pasteur:
 Fermentation is catalyzed by a vital force
contained within the yeast cells called "ferments“
• 1897: Eduard Buchner
 Rejected the vital force hypothesis.
 Nobel Prize in 1907 [Chemistry]
• Wilhelm Kühne coined the term “enzyme”
• 1926: Sumner:
 Isolated and crystallised Urease
 All enzymes are proteins
• Haldane: Enzymes catalyse by forming weak
interactions with substrate

Nomenclature:
• Based on their function: Pepsin, Lysozyme
• Based on their source: Trypsin
• By adding –ase to the substrate: Urease
• Short phrase describing their activity: DNA polymerase,
Alcohol Dehydrogenase
• IUBMB: International Union of Biochemistry and
Molecular Biology
Eg: Hexokinase
 Systematic Name: ATP: Hexose phospho-transferase
 Enzyme Commission Number [EC No.] is 2.7.1.1
 Class. Sub-Class. Sub-sub-Class. Specific Enzyme

Enzymes belong to One of These Six Classes

Most Enzymes are Proteins
• Water soluble
• Amphoteric
• Definite pI
• Monomeric or Multimeric
• The specificity of an enzyme is a result of the
intricate tertiary or quaternary structure
• But there are enzymes that are not proteins,
but RNA Ribozymes
• Eg: Peptidyl transferase, Ribonuclease P,
SnRNA

Ribozymes (Ribonucleic acid enzymes)

Many Enzymes Require Cofactors for Activity
Co-
factor
Apo-
enzyme
Holo-
enzyme
Co-enzymes: Small organic molecules
usually from vitaminsMetal Ions
Co-factors which are tightly/covalently linked are
called Prosthetic groups

Laws of Thermodynamics:
• The First Law: The total energy of a system and its
surroundings is constant.
• The Second Law: The total entropy of a system
plus that of its surroundings always increases.
• System: The matter within a defined region of
space.
• Surroundings: The matter in the rest of the
universe.
• Entropy (S): The measure of the degree of
randomness or disorder in a system.
• If heat flows from the system to its surroundings,
then the heat content is often referred to as the
Enthalpy (H)

Free energy or Gibbs free energy
• A change in the entropy of the surroundings is given by
∆Ssurroundings = -∆Hsystem /T (1)
• The total entropy change is given by the expression
∆Stotal = ∆Ssystem + ∆Ssurroundings (2)
• Substituting equation 1 into equation 2 yields
∆Stotal = ∆Ssystem - ∆Hsystem/T (3)
• Multiplying by -T gives
-T∆Stotal = ∆Hsystem - T∆Ssystem (4)
• The function –T∆S has units of energy and is referred to
as free energy or Gibbs free energy. (after Josiah
Willard Gibbs, 1878)
∆G = ∆Hsystem – T∆Ssystem (5)

Spontaneity of a reaction/process
• For a process to take place, the entropy of the
universe must increase. The total entropy will
increase if and only if
∆Ssystem > ∆Hsystem/T (6) (∆Stotal = ∆Ssystem - ∆Hsystem/T)
• i.e., if T∆S system > ∆H or, in other words, entropy
will increase if and only if
∆G = ∆Hsystem - T∆Ssystem < 0 (7)
• Thus, the free-energy change must be negative
for a process to take place spontaneously. There
is negative free-energy change when and only
when the overall entropy of the universe is
increased.

What does ∆G tell?
1. A reaction can take place spontaneously only if ∆G
is negative. (Exergonic Reaction).
2. A system is at equilibrium and no net change can
take place if ∆G is zero.
3. A reaction cannot take place spontaneously if ∆G is
positive. They requires energy (Endergonic
Reaction).
A + BC + D
4. The ∆G of a reaction depends only on the free
energy of the products minus the free energy of
the reactants. (independent of the path or
molecular mechanism).
5. The ∆G provides no information about the rate of a
reaction.

How Do Enzymes Act?
• Enzymes accelerate reactions by facilitating the
formation of the transition state
• A chemical reaction of substrate S to form product
P goes through a transition state X‡ that has a
higher free energy than does either S or P
• S  X‡  P
• Transition state :
– Transitory molecular structure that is no longer
the substrate but is not yet the product.
– It is the least-stable and most-seldom occupied
species along the reaction pathway because it
is the one with the highest free energy

How Do Enzymes Act?
• Gibbs free energy of activation
[activation energy, G‡]:
 The difference in free energy
between the transition state
and the substrate.
• Enzymes function to lower the
activation energy, i.e., they
facilitate the formation of the
transition state.
• In the transition state, the
energy barrier for the
conversion to the product is
overcome, and thus it facilitates
product formation
•When compared to a non-catalyzed reaction, Enzyme catalysed
reaction will have more molecules in the transition state, thus
increasing the rate of the reaction.

How Do Enzymes Decrease the Activation Energy?
• Chemical reactions of many types take place between
substrates and enzyme’s functional groups (specific
amino acid side chains, metal ions, and coenzymes)
• These groups may form covalent bonds with substrates
or may get transferred to the substrate, activating the
substrate
Or
• Multiple non-covalent interactions
occur between enzymes and substrates.
• Each of these weak interaction releases
energy [Binding Energy]
• Binding energy is the main source of
energy by which enzymes lower the
activation energy.
• It is also the basis for specificity of
enzymes

Active Site:
• Region that binds the substrates (and cofactor)
• Contains residues that directly participate in the making
and breaking of bonds- Catalytic Groups
• 3-dimensional cleft, or crevice, formed by groups that
come from different parts of the amino acid
• Takes up a small part of the total volume of an enzyme
• Substrates bind to active site by multiple weak attractions
• The specificity of binding depends
on the precisely defined arrangement
of atoms in an active site
• Nature of the active site has been
explained by two models
 Lock and Key model by Fischer
 Induced fit hypothesis by Koshland

• Active site:
1. Restricts the relative
motion of substrates and
orients them for reaction
[Entropy reduction]
2. Replaces water of
solvation of substrates by
own weak bonds
3. Stabilizes transition state

Ribozyme
Enzyme
Proenzyme
Isoenzyme
Isoforms
Apoenzyme
Holoenzyme
Co-enzyme
Abzyme
Allelozyme
Endozyme

Enzymes-
Mechanism and
Kinetics

Mechanisms of Catalysis:
• Catalysis by proxomity:
• When an enzyme binds substrate molecules in its active site, it creates
a region of high local substrate concentration
• Acid Base Catalysis
•General Acid Base Catalysis:
Acid groups of enzymes react with basic groups of substrates
and vice versa
•Specific Acid Base Catalysis:
Interaction between H+ and OH- ions of enzyme and substrate
• Covalent Catalysis
•Transient covalent bond occurs between enzyme and substrate
• Catalysis by strain
•Lytic enzymes bind their substrate in a conformation slightly
unfavorable for the bond that will undergo cleavage.

Chymotrypsin Shows Acid Base
and Covalent Catalysis
Covalent Catalysis
Specific
Acid Base Catalysis

Acid Base Catalysis
in HIV Protease
1. Aspartate X acts as a base to
activate a water molecule by
abstracting a proton.
2. The activated water molecule
attacks the peptide bond,
forming a transient tetrahedral
intermediate.
3. Aspartate Y acts as an acid to
facilitate breakdown of the
tetrahedral intermediate and
release of the split products by
donating a proton to the newly
formed amino group.
Subsequent shuttling of the
proton on Asp X to Asp Y
restores the protease to its
initial state.

Metal Ion Catalysis:
• A metal ion may serve as a bridge between
enzyme and substrate, increasing the binding
energy and holding the substrate in a
conformation appropriate for catalysis.
• Metals can mediate oxidation- reduction
reactions by changing their oxidation state
• Metals can form ternary complexes:
1. E-S-M Eg: Kinases; E-ATP-M
2. E-M-S Eg: Alkaline Phosphatase
3. M-E-S Eg: Pyruvate kinase
M
4. E
S

Factors Affecting the Rate of Enzyme
Catalyzed Reaction
1. Temperature
2. pH
3. Product concentration
4. Enzyme concentration
5. Substrate concentration
6. Inhibitors
7. Covalent modification
8. Induction/ Repression

Effect of 1) Temperature and 2) pH:
• Optimum temperature
• Optimum pH

3) Product Concentration
• Reaction is slowed/ stopped/ reversed when
product concentration is increased at
equilibrium
• Important in enzyme deficiencies in metabolic
pathways, as this leads to accumulation of
substrate [which is the product of preceding
pathways], thus blocking the whole pathway.
• In deficiency of E3, C accumulates, which in
turn blocks E2.
DCBA E3E2E1


4) Enzyme Concentration:
• Rate of reaction is directly proportional to enzyme
concentration when sufficient substrate is present.
• This can be used to determine the level of enzyme in
serum/plasma/tissue etc.
• Enzyme activity is measured by:
1. End point assay
 Product formation is combined with a chemical reaction
forming colored product and the intensity of color is measured
2. Kinetic assay
 Reaction is linked with another reaction requiring NAD+/NADH
 NADH, not NAD+ absorbs UV at 340 nm
 Rate of change of absorption of NADH will give the enzyme
activity
 Eg. Lactate Dehydrogenase

Units of Enzyme Activity:
• International Unit [IU]:
Amount of enzyme catalyzing the
transformation of 1 μmol of substrate into
product in 1 minute at 25 °C
• SI unit of enzyme activity is katal [Kat]
1 kat denotes conversion of 1 mol of
substrate in 1 second.
• 1 kat = 6 x 107 IU
• 1 IU = 16.7 nkat

5) Effect of Substrate Concentration:
• The effect of substrate concentration changes,
as the substrate is converted to product

• Enzyme reacts with substrate to form ES
complex
• In the initial phases of reaction, k-2 is negligible
• Three different constants can be made into
one, that is KM or Michaelis constant

Michaelis Menten Equation Deals With Enzyme
Kinetics & Substrate Concentration
• V0 = Initial rate of reaction
• Vmax = Maximum rate of reaction
• KM = Michaelis Constant
• [S] = Substrate concentration
• When KM = [S],
• KM is equal to the substrate concentration at which
the reaction rate is half its maximal value
• Unit of KM: Mole/ L

Determination of KM : Line Weaver Burk Plot/
Double Reciprocal Curve

Importance of KM
• KM is equal to the substrate concentration at
which the reaction rate is half its maximal
value
• Hallmark of an enzyme
• High KM denotes low affinity
• Different isoforms of the same enzyme have
different KM . Example: Glucokinase and
Hexokinase

6.Enzyme Inhibition
• Competitive inhibition
• Non-competitive inhibition
– Reversible
– Irreversible
• Un-competitive inhibition
• Suicide inhibition
• Allosteric inhibition

Competitive Inhibition:
• Inhibitor competes with the substrate for the
active site of the enzyme
• Diminishes the rate of catalysis by reducing the
proportion of enzyme molecules bound to a
substrate
• Inhibitor is usually a substrate analogue
• Usually reversible in presence of excess substrate

Methotrexate Competitively Inhibits
Dihyrofolate Reductase

KM ↑ and Vmax is Unchanged in
Competitive Inhibition

Many Competitive Inhibitors are Used as Drugs
• Sulfonamides inhibit Folate Synthase
• Methanol and Ethanol competes for Alcohol
Dehydrogenase
• Ibuprofen inhibits Cyclo-oxygenase
• Statins inhibit HMG CoA Reductase
• Warfarin inhibits Vitamin K Epoxide Reductase

Non-competitive Inhibition:
• Inhibitors are not substrate analogues
• Not relieved by increasing the substrate
concentration

KM is unchanged but Vmax ↓ in
Non-competitive Inhibition:

Non-competitive Inhibitors
• Irreversible
 Cyanide inhibits Cytochrome Oxidase
 Fluoride inhibits Enolase by removing Mg2+
and Mn2+
 Iodoacetate and Heavy metals bind with SH
group of enzymes
• Reversible
 Trypsin inhibitors from Ascaris
 Alpha 1 Antitrypsin in Humans

Un-competitive Inhibtion:
• Inhibitor binds to Enzyme-Substrate complex
• It cannot bind to the free enzyme, thus it
differs from Non-competitive inhibition

KM and Vmax↓ in Un-competitive
Inhibition

Suicide Inhibition [Mechanism Based
Inactivation]
• The inhibitor binds to the enzyme as a
substrate and is initially processed by the
normal catalytic mechanism.
• The mechanism of catalysis then generates a
chemically reactive intermediate that
inactivates the enzyme through covalent
modification.
• Examples:
– Allopurinol and Xanthine Oxidase
– DFMO and Ornithine Decarboxylase
– Aspirin and Cyclo-oxygenase

Allopurinol and Suicide Inhibition

Regulation of Enzyme Activity:
• Quantitative
 At the level of synthesis and degradation
 Mainly under genetic control [Induction/
Repression]
 Slow
• Qualitative [Catalytic efficiency]
 Allosteric modification
 Covalent modification
 Proteolytic activation

Allosteric Modification
• Has an allosteric site, which is different from
active site
• The regulatory molecule binds to the allosteric
site
• Regulatory molecule is not a structural analogue

Allosteric Regulation:
• Usually regulate metabolic pathways
• Key enzyme or Rate limiting enzyme
• Eg: Aspartate Transcarbamoylase regulated by
CTP
• Binding/Dissociation of one regulatory
molecule stimulates Binding/Dissociation of
others [Co-operativity]

Kinetics of Allosteric Enzymes:
• Allosteric enzymes does not obey Michaelis
Menten Kinetics
• It can alter KM or Vmax or both
K series enzymes V series enzymes

Phosphorylation/ Dephosphorylation is
the Most Common Covalent Modification
• Serine, Threonine and Tyrosine residues
• Can activate/ inhibit enzyme activity

Limited Proteolysis Can Activate Zymogens:

Application of Enzymes:
1. Diagnostic
2. Therapeutic
• Streptokinase : Thrombolysis in MI
3. Analytical
• Glucose peroxidase : Glucose
• Urease : Urea
• Uricase : Uric acid
• Horse radish peroxidase in ELISA
4. Genetic engineering
• Restriction endonuclease : Genetic Engineering
• Taq DNA polymerase : PCR
5. Industrial
• Rennin : Cheese preparation
• α-amylase : Food industry

Enzymes of Diagnostic Significance:
1. Functional plasma enzymes:
• Eg: LPL, proenzymes of blood coagulation and
clot dissolution
2. Non-functional plasma enzymes:
• No physiological function in blood
• Increased level suggests tissue destruction
• Diagnostic & prognostic value
Exocrine enzymes
 Diffuse into plasma
e.g. Pancreatic amylase, lipase, prostatic ACP
True intracellular enzymes
 Normally absent from circulation
e.g. Lactate dehydrogenase, aminotransferases

Entry of Enzymes Into the Blood
1. Rate of enzyme leak from cells
• Plasma membrane integrity depends upon
energy production which depend upon O2 -
↓oxygenated blood perfusion. E.g. MI
• Direct attack on cell membrane by virus or
organic chemicals. E.g. Liver
• Skeletal muscle - poor perfusion, direct
trauma to muscle
• Necrosis of cell

Entry of Enzymes Into the Blood
2. Altered rate of enzyme production
• Proliferation of particular type of enzyme
producing cells [↑ rate of cellular turnover ]
E.g: ↑Prostatic acid phosphate
• ↑ rate of enzyme synthesis due to ↑ activity
of a particular enzyme producing cells-
E.g: Osteoblast – ALP
• Enzyme induction-
E.g: ↑GGT due to administration of
barbiturates, phenytoin
3. Rate of clearance
• Due to receptor mediated endocytosis by RE
cells. [amylase by kidney]

Characteristics of Markers:
• High concentration in that tissue/organ
• Not found in other tissues.
• Released rapidly and completely.
• Released in direct proportion to the damage.
• Provide a convenient diagnostic time window.
• Determining Factors
 Size
 Solubility
 Specificity
 Specificity for irreversible injury
 Cellular localization
 Release ratio
 Detectability
 Clearance

Enzymes in Diagnosis:
Enzyme
Normal
Level IU/L
Clinical Use
Alanine Amino Transferase [ALT] 7-41 Liver Diseases
Aspartate Amino Transferase [AST] 12-38
Liver Diseases
Myocardial Infarction
Amylase 20-96 Pancreatitis
Lipase 0-1 Pancreatitis
Choline Esterase 5-12
Organophosphorus
Poisoning
Creatinine Kinase- Total [CK] 39-238
Muscle Disorders

Enzymes in Diagnosis:
Enzyme
Normal
Level IU/L
Clinical Use
γ Glutamyl Transferase [GGT] 9-58 Alcoholism
Alkaline Phosphatase 3-96
Bile Duct Obstruction
Liver Diseases
Bone Diseases
Acid Phosphatase 3-12 Ca Prostate
5’ Nucleotidase 0-11 Bile Duct Obstruction
Lactate Dehydrogenase [LDH] 115-220
Hemolysis

Isoenzymes or Isozymes:
• Physically distinct forms of same enzyme
• Catalyze similar reaction
• Differ from each other structurally,
electrophoretically and immunologically
• Coded by distinct genes that can be at
different loci or can represent different
alleles at the same locus
• Genes encoding isoenzymes- can be on same
chromosome or on different chromosomes

Isoenzymes of CK
Isozyme
Molecular
Form
Tissue
CK1 BB Brain
CK2 BM Heart
CK3 MM Muscle

Isozymes of LDH
Number 1 to 5 are assigned on
the basis of anodal migration
during electrophoresis, 1 being
the first or fastest and 5 the
slowest
Isozyme
Molecular
Form
Tissue
LDH1 H4 Heart
LDH2 H3M1 RBC
LDH3 H2M2 Brain
LDH4 H1M3 Liver
LDH5 M4 Muscle

LDH in Myocardial Infarction:
• Rises within 8-12 h, peak at 28-48 h, and
remain elevated for >7 days.
• Usually 3-4 fold rise
• LDH-1 has an increased tissue specificity.
• Normally LDH-2 > LDH-1
• Normally LDH-1/LDH-2 is < 0.76
• Ratio > 0.76 suggestive of AMI, value >1.0
improves specificity (Flipped ratio).
• Persistent abnormality in LDH-1/LDH-2 ratio
indicates reinfarction.
• Intravascular or extravascular haemolysis ↑
LDH1 and LDH2 levels.

Isoforms:
• Coded by same gene
• Catalyze same reaction
• Formed by post translational modification
• Can be separated by electrophoresis
• E.g:
• CK-33 – gene product
• CK-32 – one lysine residue removed
• CK-31 – both lysine removed
• CK-22 – gene products
• CK-21 – lysine residue removed

Biochemical Markers of MI
A. Enzymatic:
• Creatine Kinase (CK)
• Lactate Dehydrogenase (LDH)
• Asparatate Amino Transferase (AST)
B. Non-enzymatic:
1. Cytoplasmic
• Myoglobin
• Heart Fatty Acid Binding proteins
2. Non-cytoplasmic
• Myosin Fragments
• Troponins

Aspartate Aminotransferase
• L-Aspartate + α-Ketoglutarate ↔ Oxaloacetate + L-Glutamate
• Highest concentration in heart, also present in liver,
skeletal muscle, kidneys and brain.
Diagnostic significance
• Serum level increase whenever disease process affect
liver cell integrity.
• Myocardial infarction (rises after 6-8 hrs of onset of
chest pain, peak 18-24 hrs, return to normal 4-5 day)
• Extent of myocardial damage appears to parallel peak
values reached from release from myocardium after
infarction

Alanine Aminotransferase
• L-Alanine + α-Ketoglutarate ↔Pyruvate + L-Glutamate
• Found in many tissues, with high levels in Liver
• Confined mainly to evaluation of hepatic disorders.
• Higher elevation in hepatocellular disorder than
extra or intrahepatic obstructive disorders.

γ-Glutamyl Transferase [GGT]
Glutathione +amino acid → glutamyl peptide + L-cysteinyl glycine
• Found in kidney, prostate, pancreas and liver
• Involved in peptide and protein synthesis.
• Regulation of tissue glutathione level.
• Transport of amino acid across cell membranes.
• Hepatobiliary disorder
• Enzyme inducing drugs
• Alcoholics and heavy drinkers
• Differentiating – liver, skeletal disorder and pregnancy
• Hepatobiliary involvement in adolescents.

Amylase:
• Starch, glycogen → glucose, maltose, dextrins
• MW 55,000 - 60,000 Dalton
• Only plasma enzyme found in urine
• Greatest concentration in pancreas- P type
• Salivary amylase – S type
• Macroamylase – S type and Ig A, Ig G or other
cannot filter through kidney
• Investigation of pancreatic function
• Acute pancreatitis:
– Begins to rise by 2-12 hours, peak by 24 hours,
return to normal by 3-5 days

Lipase:
• Triglyceride + H2O ↔ Glycerol + Fatty acids
• MW: 54,000 Dalton
• In pancreas, 100 fold greater than other tissues
• Lingual gland, gastric, pulmonary and intestinal
mucosa
• Acute pancreatitis, increases in 4-8 hours, peaks in
24 hour and returns to normal in 8-14 days.

Alkaline Phosphatase- ALP
• Intestine, liver, bone, spleen, placenta and kidney
• LIVER
– Extrahepatic obstruction (>3 fold)
– Intrahepatic obstruction of bile flow ( up to 2.5 UNL)
– Parenchymal liver disease (up to 3 fold)
• BONE
– Elevation when involvement of osteoblasts
• PREGNANCY
– 2-3 times in third trimester
• INTESTINE
– Disease of digestive tract and cirrhosis
• REGAN AND NAGAO
– Referred to carcinoplacental ALP due to similarity to
placental isoenzyme

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