Enzymes

• Enzymes are biological catalysts, mostly proteins, that speed up chemical reactions in the
body without being consumed.
• They ensure that specific pathways are followed by selectively binding to substrates.
Enzyme Classification & Nomenclature
Enzymes are classified by the Enzyme Commission (EC) of the International Union of Biochemists
and Molecular Biologists (IUBMB) into six major classes, based on the type of reaction catalyzed.
Each enzyme is assigned:
• A systematic name (describes the reaction),
• A common/trivial name (e.g., trypsin, pepsin),
• An EC number (four-part identifier, e.g., EC 2.7.1.2).

Six Major Enzyme Classes

EC Class Function
Number
EC 1 Oxidoreductases Catalyze oxidation-reduction reactions (e.g., dehydrogenases,
oxidases).
EC 2 Transferases Transfer functional groups from one molecule to another (e.g.,
kinases, aminotransferases).
EC 3 Hydrolases Catalyze hydrolysis reactions (e.g., proteases, phosphatases).
EC 4 Lyases Break C-C, C-O, C-N bonds without hydrolysis or oxidation (e.g.,
decarboxylases, aldolases, synthases).
EC 5 Isomerases Rearrange atoms within a molecule to form isomers (e.g.,
mutases, racemases).
EC 6 Ligases Form new bonds (C-C, C-O, C-N, C-S) using ATP (e.g., synthetases,
carboxylases).

Important Naming Notes

Term Meaning
Synthetase Requires ATP to form new bonds (ligase class).
Synthase Forms new bonds without ATP (lyase class).
Phosphatase Removes phosphate using water (hydrolase).
Phosphorylase Adds phosphate using Pi (inorganic phosphate).
Dehydrogenase Uses NAD⁺/FAD as electron acceptors in redox reactions.
Oxidase Uses O₂ as the electron acceptor without incorporating oxygen.
Oxygenase Incorporates one or both oxygen atoms into the substrate.

Properties of Enzymes
1. Protein Catalysts:
Enzymes are mostly proteins (except ribozymes, which are RNA-based) that speed up
reactions without being consumed.
2. Active Site:
A specific pocket on the enzyme where substrate binds, forming the enzyme-substrate (ES)
complex. This often causes a conformational change (induced fit), leading to product
formation and release.
3. Catalytic Efficiency:
Enzymes accelerate reactions 10³–10⁸ times faster than uncatalyzed ones. The turnover
 number (kcat) refers to the number of substrate molecules converted to product per enzyme
per second.
4. Specificity:
Enzymes act on specific substrates and catalyze specific types of reactions.
5. Holoenzymes:
o Holoenzyme = Apoenzyme (protein part) + non-protein component.
o Cofactors: Inorganic (e.g., metal ions like Zn²⁺).
o Coenzymes: Organic molecules (often vitamin-derived).
▪ Cosubstrates: Transiently bound (e.g., NAD⁺).
▪ Prosthetic groups: Tightly bound (e.g., FAD, biotin).
o Metalloenzymes: Enzymes with tightly bound metal ions.
6. Regulation:
Enzyme activity is modulated according to cellular needs.
7. Localization:
Enzymes are compartmentalized in cells, which helps isolate reactions and organize
metabolic pathways.

MECHANISM OF ENZYME ACTION

Enzymes accelerate reactions by:
1. Lowering the free energy of activation (energetically favorable pathway).
2. Utilizing active site chemistry to catalyze substrate conversion.
A. Energy Changes During Enzyme-Catalyzed Reactions
1. Free Energy of Activation (ΔG‡):
o All reactions have an energy barrier between reactants and products.
o This energy (activation energy) is required to reach the transition state (T*), a high-
energy intermediate.
o Enzymes reduce this barrier, making reactions faster.
2. Rate of Reaction:
o Only molecules with energy ≥ ΔG‡ can form products.
o Enzymes increase the number of such molecules by lowering ΔG‡, thus increasing
reaction speed.
3. Alternate Pathway:
o Enzymes offer a different reaction route with a lower ΔG‡.
o They do not change the reactant/product energy or equilibrium—just the speed to
reach it.
B. Chemistry of the Active Site
The active site is a dynamic region, precisely shaped to:
• Bind substrates (often via hydrogen bonds, ionic interactions, hydrophobic effects).
• Stabilize the transition state, increasing conversion rate.
Binding Models:
1. Lock-and-Key Model:
o Active site has a fixed shape complementary to the substrate.
o Substrate fits exactly like a key in a lock.
2. Induced-Fit Model:
o Active site molds around the substrate upon binding.
o Better explains enzyme flexibility and specificity.
Catalytic Mechanisms Used by Enzymes
Enzymes use combinations of these strategies:
 1. Catalysis by Proximity:
o Enzyme binds multiple substrates close together and in correct orientation.
o Increases local concentration and frequency of effective collisions.
o Speeds up reactions up to 1000x.
2. Acid-Base Catalysis:
o Uses acidic or basic side chains (e.g., histidine, glutamate).
o Two types:
▪ Specific: Involves only H⁺ or OH⁻.
▪ General: Involves other acids/bases in the active site or solution.
o Aids proton transfer, stabilizing charged transition states.
3. Catalysis by Strain (Transition State Stabilization):
o Enzyme distorts the substrate into a high-energy, transition-like state.
o Weakens specific bonds, making them easier to break.
o Common in lytic reactions (bond cleavage).
4. Covalent Catalysis:
o Enzyme forms a temporary covalent bond with substrate.
o A new, lower energy intermediate forms, speeding up the reaction.
o Often seen in group transfer reactions.
o Common residues: serine, cysteine, histidine.
o Follows ping-pong mechanism:
▪ First substrate binds → product released.
▪ Second substrate binds → final product formed.

Michaelis-Menten Kinetics
A. Reaction Model
Michaelis and Menten proposed a model for enzyme-catalyzed reactions:
• An enzyme (E) binds reversibly with a substrate (S) to form an enzyme-substrate (ES)
complex.
• The ES complex then breaks down into product (P), regenerating the free enzyme.
• E + S ⇌ ES → E + P

B. Michaelis-Menten Equation
The Michaelis-Menten equation describes how the initial reaction velocity (V₀) varies with
substrate concentration ([S]):

Assumptions in the derivation:

1. [S] >> [E]: Substrate concentration is much greater than enzyme, so only a small
fraction of substrate is bound at a time.
2. Steady-State Assumption: The formation and breakdown of the ES complex reach a
steady state (constant [ES]).
 3. Initial Velocity (V₀): Measurements are made early in the reaction, when the product
concentration is negligible and the reverse reaction is insignificant.

C. Key Conclusions from Michaelis-Menten Kinetics

1. Km (Michaelis Constant)
• Indicates the affinity between enzyme and substrate.
• Defined as the substrate concentration at which V₀ = ½ Vmax.
• Low Km = High affinity (less substrate needed to reach half-max velocity).
• High Km = Low affinity (more substrate required).
2. Vmax (Maximum Velocity)
• The maximum rate at which the enzyme can convert substrate to product.
• Achieved when all enzyme active sites are saturated with substrate.
• Proportional to enzyme concentration.
3. Velocity vs. Enzyme Concentration
• Reaction rate increases linearly with enzyme concentration at all [S].
• If [E] is halved, V₀ and Vmax are also halved.
4. Order of Reaction
• When [S] << Km → reaction is first-order (rate ∝ [S]).
• When [S] >> Km → reaction is zero-order (rate independent of [S]).

D. Lineweaver-Burk Plot (Double-Reciprocal Plot)

• A linear transformation of the Michaelis-Menten equation.
• Plots 1/V₀ vs 1/[S], yielding a straight line:

• X-intercept = –1/Km
• Y-intercept = 1/Vmax
• Useful for determining Km, Vmax, and types of enzyme inhibition.

Factors Affecting Enzyme Reaction Velocity

A. Substrate Concentration
• As [S] increases, velocity (V) rises until Vmax is reached.
• At high [S], enzymes become saturated, and further increase in [S] doesn’t affect the
rate.
• The resulting curve is hyperbolic for enzymes obeying Michaelis-Menten kinetics.
• Allosteric enzymes show a sigmoidal curve, reflecting cooperative substrate binding.

B. Enzyme Concentration
• Reaction rate is directly proportional to enzyme concentration.
• Doubling enzyme concentration doubles the rate (and Vmax), regardless of [S].
C. Temperature
1. Rate increases with temperature up to an optimum (~35–40°C in humans), as more
molecules overcome the activation energy.
2. Beyond optimum, high temperature causes enzyme denaturation, reducing activity.
o Enzymes from thermophiles can function optimally at ~70°C.

D. pH
1. Affects ionization of active site residues: Correct protonation states are essential for
catalysis.
o E.g., protonated –NH₃⁺ may be required; high pH deprotonates and inhibits
function.
2. Extremes in pH lead to enzyme denaturation due to disruption of ionic bonds.
3. Each enzyme has an optimal pH, reflecting its physiological environment.
o Pepsin: Optimum at pH 2 (stomach)
o Most human enzymes: Optimum near pH 7
INHIBITION OF ENZYME ACTIVITY
Enzyme inhibitors are substances that reduce or halt the activity of enzymes, impacting the
velocity of enzyme-catalyzed reactions. Inhibitors are broadly categorized into irreversible and
reversible types based on how they interact with enzymes.
A. IRREVERSIBLE INHIBITION
• Covalent binding with enzyme’s active site or essential groups.
• Permanent inactivation of enzyme activity.
• Example: Organophosphates, which bind irreversibly to acetylcholinesterase.
B. REVERSIBLE INHIBITION
Reversible inhibitors bind noncovalently and can dissociate, allowing enzyme activity to be
restored. They include:
1. Competitive Inhibition
• Mechanism: Inhibitor competes with substrate for the same active site.
• Effect on Km: Increases (apparent Km) – higher substrate needed to achieve ½ Vmax.
• Effect on Vmax: Unchanged – high [S] can overcome inhibition.
• Lineweaver-Burk Plot: Intersects at y-axis (1/Vmax same), slope increases.
• Example:
o Statins (e.g., atorvastatin) inhibit HMG-CoA reductase to reduce cholesterol.
• Clinical Drugs:
o Methotrexate – anticancer (inhibits dihydrofolate reductase)
o Sulphonamides – antibiotics (inhibit folate synthesis)
o Alpha-methyl dopa – antihypertensive
o Dicoumarol – anticoagulant

2. Noncompetitive Inhibition
• Mechanism: Inhibitor binds to a site other than the active site, on either the free
enzyme or ES complex.
• Effect on Km: Unchanged – substrate binding not affected.
• Effect on Vmax: Decreased – active enzyme amount effectively reduced.
 • Lineweaver-Burk Plot: Intersects at x-axis (Km same), y-intercept increases.
• Example:
o Lead inhibits ferrochelatase (heme synthesis).
o Insecticides inhibit acetylcholinesterase.

3. Uncompetitive Inhibition
• Mechanism: Inhibitor binds only to ES complex (not to free enzyme).
• Effect on Km: Decreased – increases apparent affinity of enzyme for substrate.
• Effect on Vmax: Decreased – reduces overall enzyme turnover.
• Lineweaver-Burk Plot: Parallel lines – both Km and Vmax decrease.
• Example:
o Phenylalanine inhibits placental alkaline phosphatase (Regan isoenzyme).
• Works best at high substrate concentrations.

Comparison Table
Feature Competitive Noncompetitive Uncompetitive
Binding Site Active site Allosteric site ES complex only
Km ↑ (increases) No change ↓ (decreases)
Vmax No change ↓ (decreases) ↓ (decreases)
Reversed by [S]? Yes No No
Lineweaver-Burk Intersect y-axis Intersect x-axis Parallel lines

D. Enzyme Inhibitors as Drugs

Many clinically used drugs function as enzyme inhibitors:
• Penicillin: Inhibits transpeptidase, blocking bacterial cell wall synthesis.
• ACE inhibitors: Block angiotensin-converting enzyme, reducing blood pressure.
o Examples: Captopril, Enalapril, Lisinopril
• β-lactam antibiotics: Target bacterial enzymes, crucial for antibacterial therapy.

Regulation of Enzyme Activity

Proper regulation of enzyme activity is crucial for coordinating metabolic processes. Enzyme activity
can be modulated in three main ways:
A. Allosteric Regulation
• Allosteric enzymes are usually multisubunit enzymes that catalyze committed steps in
pathways.
• They are regulated by effectors (modifiers) that bind noncovalently at sites other than the
active site.
Types of Effectors:
1. Homotropic Effectors:
o The substrate itself acts as the effector.
o Binding enhances activity at other sites (cooperativity).
o Results in a sigmoidal velocity vs. substrate ([S]) curve.
2. Heterotropic Effectors:
o The effector is different from the substrate.
 o Example: Feedback inhibition, where the end product inhibits an early step.
o Example: Citrate inhibits PFK-1, a glycolytic enzyme.
Additional Mechanisms:
• Feedforward Activation: A metabolite activates a later enzyme.
o Example: Fructose-1,6-bisphosphate activates pyruvate kinase.
• Reciprocal Regulation: One effector can stimulate one enzyme and inhibit another.
o Example: AMP activates PFK-1 and inhibits fructose-1,6-bisphosphatase.

B. Covalent Modification
• Involves phosphorylation/dephosphorylation of enzymes at serine, threonine, or tyrosine
residues.
• Catalyzed by:
o Protein kinases (add phosphate from ATP)
o Phosphatases (remove phosphate)
Effect on Enzyme Activity:
• Can activate or inactivate enzymes depending on the enzyme.
o Example:
▪ Phosphorylation activates glycogen phosphorylase.
▪ Phosphorylation inactivates glycogen synthase.

C. Enzyme Synthesis (Induction and Repression)

• Regulates amount of enzyme by altering synthesis or degradation rates.
• Induction: Increased synthesis (e.g., insulin increases glucose-metabolizing enzymes).
• Repression: Decreased synthesis.
• These changes are slower (hours to days) compared to allosteric or covalent regulation
(seconds to minutes).

Enzymes in Clinical Diagnosis

Plasma Enzymes and Their Role in Diagnostics: Plasma enzymes are classified into two main
categories:
1. Active Enzymes Secreted into the Blood:
o A small group of enzymes that are secreted by specific cells (e.g., liver secretes
zymogens, inactive precursors of blood coagulation enzymes).
2. Enzymes Released Due to Cell Turnover:
o A larger number of enzymes are released from cells during their normal
turnover. These enzymes typically function intracellularly and have no
physiological role in the plasma. The level of these enzymes remains constant
in healthy individuals as a balance between enzyme release and removal.
o When tissue damage occurs, enzyme levels in the plasma increase, serving as
markers for tissue injury.
Alterations in Plasma Enzyme Levels in Disease:
• Elevated plasma enzyme levels are often indicative of tissue damage. For example, the
enzyme alanine aminotransferase (ALT), which is abundant in the liver, rises in plasma
levels in cases of liver damage (e.g., hepatitis).
 • Diagnostic Value: Some enzymes are tissue-specific, and their increased levels in
plasma suggest damage to the corresponding organ. Enzymes with broader tissue
distribution are less specific in identifying the tissue of origin.
Isoenzymes:
• Isoenzymes are different forms of the same enzyme that catalyze the same reaction
but exist in different tissues and have variations in their structure, amino acid
composition, electrophoretic mobility, and thermostability.
Types of Isoenzymes:
1. Heteroenzymes: Isoenzymes from different species or tissues that catalyze the same
reaction but differ chemically (e.g., yeast vs. animal alcohol dehydrogenase).
2. Tissue-Specific Isoenzymes: Isoenzymes with similar activities but differing in heat
stability and susceptibility to inhibitors (e.g., alkaline phosphatase from different
organs).
3. Electrophoresis: Isoenzymes can be separated based on their charge differences using
electrophoresis, aiding in diagnostics.

Clinical Significance of Isoenzymes:

Lactate Dehydrogenase (LDH):
• LDH catalyzes the conversion of pyruvic acid to lactic acid. It exists in five isoenzymes:
LDH1, LDH2, LDH3, LDH4, and LDH5. Each isoenzyme has a unique combination of H
and M subunits and varies in different tissues.
• Clinical Significance:
o LDH1 and LDH2 are elevated in myocardial infarction (MI), while LDH5 and
LDH4 are associated with liver and muscle damage.
o Flipped pattern (LDH2 > LDH1) is indicative of MI.
o The identification of LDH isoenzymes aids in distinguishing between myocardial
damage and liver or muscle injury.
Creatine Kinase (CK):
• CK exists in three isoenzymes: CK-MM (skeletal muscle), CK-MB (heart muscle), and
CK-BB (brain). It is absent in RBCs, liver, and kidneys but present in small amounts in
other tissues like the adrenal glands and thyroid.
• Clinical Significance:
o CK-MB is elevated in myocardial infarction and is used as a diagnostic marker.
o CK levels rise significantly within 4-6 hours of MI and peak at 24 hours.
o Troponins (cTnI, cTnT) are also key biomarkers for MI, with cTnI being highly
sensitive and specific for cardiac damage.
Enzymes in Medicine:
Enzymes and their inhibitors are also used as therapeutic agents for treating various diseases:
• Fibrinolytic Enzymes: Urokinase and streptokinase are used for dissolving blood clots
by activating plasminogen.
• Protein Digesting Enzymes: Trypsin, chymotrypsin, and pepsin are used for wound
healing and digestive disturbances.
• Cancer Treatment: Asparaginase is used to halt the growth of cancer cells by digesting
asparagine, which is essential for cancer cell proliferation.
• Antihypertensive Treatment: ACE inhibitors (e.g., captopril) are used for managing
hypertension by inhibiting the enzyme that converts angiotensin I to angiotensin II, a
potent vasoconstrictor.
• Other Therapeutic Enzymes:
o Thrombin: Used to stop bleeding during surgeries.
o Kallikrein: Increases kinins, which are vasodilators, helping improve blood flow.
o Hyaluronidase: Used in the treatment of sprains and thrombophlebitis.
These enzymes and inhibitors are powerful tools in both diagnostics and treatment, playing
key roles in modern medicine.