06 Enzymes

Introduction

This lesson covers one of the most fundamental topics in biochemistry: enzymes and enzymology. Enzymes are the molecular machines that make life possible — they catalyze virtually every chemical reaction in living cells, with extraordinary speed and precision. Understanding how enzymes work, how their activity is measured, and how it is regulated is essential for understanding metabolism, pharmacology, and the molecular basis of disease.

This lesson is organized as follows:

Protein–Ligand Interactions (the basis of enzyme function)
What Are Enzymes?
Catalysis and Activation Energy
The Active Site and Enzyme–Substrate Interaction
Models of Enzyme–Substrate Binding
Enzyme Specificity
Enzyme Kinetics (Michaelis–Menten)
Factors Affecting Enzyme Activity
Enzyme Inhibition
Regulation of Enzyme Activity
Zymogens and Protease Cascades

1. The Reversible Binding of a Ligand to a Protein

Before studying enzymes specifically, it is important to understand the general principle of protein–ligand interactions, since enzyme catalysis is built on this foundation.

A ligand is any molecule that binds reversibly to a protein (including other proteins, small molecules, ions, etc.).
Binding occurs at a specific location on the protein called the binding site, which is structurally complementary to the ligand in terms of size, shape, charge, and hydrophobic/hydrophilic character.
A given protein may have multiple binding sites for different ligands simultaneously.
The binding is typically transient — this reversibility is critical for life, allowing organisms to respond rapidly and flexibly to environmental and metabolic changes.

Induced Fit During Binding

When a ligand binds to a protein, the protein may undergo a conformational change that makes the binding site even more complementary to the ligand, enabling tighter binding. This structural adaptation is called induced fit. In multi-subunit proteins, a conformational change in one subunit can propagate to affect other subunits — a phenomenon central to allosteric regulation (discussed later).

2. What Are Enzymes?

The word enzyme comes from the Greek en zymos, meaning “leavened” (as in yeast). In 1850, Louis Pasteur observed that the fermentation of sugar to alcohol was catalyzed by “ferments,” proposing the theory of vitalism — the idea that living organisms contained special vital forces. Later work by biochemists showed these ferments were proteins.

Key Properties of Enzymes

Almost all enzymes are proteins (globular proteins). Notable exceptions are ribozymes — catalytic RNA molecules, discovered in 1982.
They have extraordinary catalytic power, often exceeding that of synthetic or inorganic catalysts by 5 to 17 orders of magnitude.
They show high specificity for their substrates.
They function under mild physiological conditions (aqueous solution, near-neutral pH, body temperature).
Their activity depends on the integrity of their native protein conformation — disrupting primary, secondary, tertiary, or quaternary structure destroys activity.
They may require cofactors: metal ions (Fe²⁺, Mg²⁺, Mn²⁺, Zn²⁺, Cu²⁺) or organic molecules (vitamins/coenzymes).

Cofactors and Coenzymes

Ion	Example Enzyme
Cu²⁺	Cytochrome oxidase
Fe²⁺/Fe³⁺	Cytochrome oxidase, catalase, peroxidase
Mg²⁺	Pyruvate kinase
Zn²⁺	Various enzymes

Coenzymes are complex organic or metalloorganic molecules that act as transient carriers of specific functional groups (e.g., NAD⁺, FAD, CoA).

Classification of Enzymes

By international agreement, enzymes are classified into six classes based on the type of reaction they catalyze:

Class No.	Class Name	Reaction Type
1	Oxidoreductases	Transfer of electrons (hydride ions or H atoms)
2	Transferases	Group transfer reactions
3	Hydrolases	Hydrolysis (transfer of functional groups to water)
4	Lyases	Cleavage of C–C, C–O, C–N bonds by elimination or addition to double bonds
5	Isomerases	Transfer of groups within molecules to yield isomers
6	Ligases	Formation of C–C, C–S, C–O, C–N bonds coupled to cleavage of ATP

3. Catalysis and Activation Energy

What Is a Catalyst?

A catalyst is a substance that:

Increases the reaction rate without being consumed or permanently altered.
Does not change the equilibrium of the reaction.
Provides an alternative reaction pathway with a lower activation energy (ΔG‡).

Energy Diagrams

Imagine a reaction: S → P (substrate to product).

Without a catalyst, the reaction must overcome a high energy barrier (the activation energy). With an enzyme:

The enzyme provides an alternative pathway where the energy barrier is significantly lower.
The net energy released (the thermodynamics of the reaction: ΔG°) is unchanged — enzymes do not make unfavorable reactions favorable; they only speed up reactions that are already thermodynamically possible.

Diagram concept: A reaction coordinate diagram shows: reactants → transition state (energy peak) → products. Without enzyme, the transition state peak is very high. With enzyme, the peak is much lower. The overall energy difference between reactants and products remains the same.

Transition States and Reaction Intermediates

In the enzyme-catalyzed reaction:

E + S ⇌ [ES] ⇌ [EP] ⇌ E + P

ES = enzyme–substrate complex
EP = enzyme–product complex
The transition state is a fleeting molecular moment at which bond breakage and bond formation are equally likely — decay to substrate or to product are equi-probable.
ES and EP can be considered transient intermediates.
For many enzymes, several steps may have similar activation energies, meaning they are all partially rate-limiting.

4. The Active Site and Binding Energy

The Active Site

Enzyme-catalyzed reactions occur within a specialized region called the active site — a pocket or cleft on the enzyme surface.
The active site is lined with amino acid residues whose side chains bind the substrate and participate in catalysis.
The molecule bound and acted upon in the active site is called the substrate.

Binding Energy (ΔG_B)

The interaction between enzyme and substrate in the ES complex is mediated by weak, noncovalent interactions:

Hydrogen bonds
Ionic (electrostatic) interactions
Hydrophobic forces
Van der Waals interactions

The formation of these weak interactions releases a small amount of free energy called binding energy (ΔG_B). This is a major source of free energy used by enzymes to lower activation energies.

How Binding Energy Lowers Activation Energy

Some weak interactions form in the ES complex, but the fully complementary interactions only form when the substrate reaches the transition state.
The active site is structured to optimize interactions with the transition state, not just the ground-state substrate.
The binding energy released by forming these interactions partially offsets the energy required to reach the transition state.
Net result: ΔG‡(net) = ΔG‡(uncatalyzed) − ΔG_B

Analogy: Imagine using magnets to illustrate this concept: an enzyme that is perfectly complementary only to its substrate (like a magnet perfectly fitted to a flat metal plate) would bind the substrate tightly but release no extra energy upon reaching the transition state — providing no catalytic benefit. The catalytic power comes specifically from preferential stabilization of the transition state.

5. Models of Enzyme–Substrate Binding

5.1 The Lock and Key Model (Emil Fischer, 1894)

The earliest model proposed that enzymes are structurally complementary to their substrates — they fit together like a lock and key:

The enzyme (lock) has a rigid, preformed active site.
The substrate (key) fits precisely into the active site.
After catalysis, the products leave and the enzyme is available again.

Limitation: This model is overly rigid. An enzyme that is perfectly complementary to its substrate would bind the substrate tightly but would not necessarily stabilize the transition state — making it a poor catalyst.

5.2 The Induced Fit Model (Daniel Koshland, 1958)

This model corrects the lock-and-key hypothesis:

The enzyme’s structure is flexible, not rigid.
The active site has an adjusted shape that changes upon substrate binding.
Binding of the substrate induces a conformational change in the enzyme that maximizes complementary interactions in the transition state.
This increases the range of substrate specificity and improves catalysis.

Classic Example — Hexokinase:

Hexokinase catalyzes: Glucose + ATP → Glucose-6-phosphate + ADP
The enzyme exists in open and closed forms.
Glucose binding induces a conformational change to the closed form, which positions catalytic residues correctly and excludes water (preventing wasteful ATP hydrolysis).

Key Principle: The active site is complementary to the transition state, not to the ground-state substrate. Enzymes stabilize the transition state, which is why they are such powerful catalysts.

6. Enzyme Specificity

Enzymes display remarkable specificity. There are three major types:

6.1 Absolute Specificity

One enzyme acts on only one substrate.

Example: Urease decomposes only urea.
Example: Arginase cleaves only arginine.

6.2 Relative Specificity

One enzyme acts on different substrates that share the same bond type.

Example: Trypsin cleaves different proteins at the carboxyl side of lysine or arginine residues.

6.3 Stereospecificity

Some enzymes can only catalyze the transformation of substrates in a specific geometric configuration (D or L; cis or trans).

Example: L-Lactate dehydrogenase acts only on L-lactate (not D-lactate).
The enzyme–substrate interaction involves a three-point attachment that distinguishes mirror images, even though the two forms appear chemically identical in solution.

Diagram concept: Imagine L-lactate binding to the enzyme at three specific points (the hydroxyl group, the carboxyl group, and the methyl group) in a precise orientation. The mirror image (D-lactate) cannot achieve the same three-point attachment — hence no reaction.

7. Enzyme Kinetics

7.1 Defining Enzyme Activity

Activity (IU — International Unit): The amount of enzyme that converts 1 µmole of substrate to product per minute under defined conditions (25°C, pH 7.4).
- Example: 10 IU = 10 µmol/min
Specific Activity: Enzymatic activity (IU) per milligram of total protein in the preparation.
- Example: 10 µmol/min/mg protein = 10 IU/mg protein
- Specific activity increases as a preparation becomes more pure (fewer contaminating proteins).

7.2 Initial Velocity (V₀) and Substrate Concentration

In a typical enzyme experiment:

[E] (enzyme concentration) is held constant and is very small (nanomolar range).
[S] (substrate concentration) is varied and is typically 5–6 orders of magnitude higher than [E].
We measure the initial velocity (V₀) — the rate of product formation at the very beginning of the reaction, before significant [S] depletion occurs.

The initial rate equation:

V₀ = k[S]

At early time points, [S] is in large excess and rate depends primarily on [S].

7.3 Saturation Kinetics and the Michaelis–Menten Equation

When V₀ is plotted versus [S] at constant [E], a hyperbolic curve results:

At low [S]: V₀ increases nearly linearly with [S] (enzyme active sites are mostly empty).
At intermediate [S]: V₀ increases by smaller increments as more active sites become occupied.
At high [S]: V₀ reaches a plateau — Vmax — where essentially all enzyme molecules are saturated with substrate (all active sites occupied).

Michaelis–Menten Equation (derived by Michaelis and Menten in 1913):

$V_{0} = \frac{V _{ma x} \cdot [ S ]}{K _{m} + [ S ]}$

Where:

V₀ = initial reaction velocity
Vmax = maximum velocity (when enzyme is fully saturated)
[S] = substrate concentration
Km = Michaelis constant

Limiting cases:

When [S] << Km: V₀ ≈ (Vmax × [S]) / Km → linear dependence on [S]
When [S] >> Km: V₀ ≈ Vmax → rate is constant, independent of [S]

7.4 The Michaelis Constant (Km)

When V₀ = ½ Vmax:

Km = [S] at which V₀ = ½ Vmax

Biological meaning of Km:

Km is an approximation of the substrate concentration required for half-maximal activity.
Smaller Km → greater affinity of the enzyme for its substrate (enzyme reaches half-maximal velocity at a lower [S]).
Larger Km → lower affinity (requires more substrate to reach half-maximal activity).
Km provides an approximation of the physiological [S] for many enzymes in vivo.

Example — Hexokinase (brain):

Substrate	Km (mM)
ATP	0.4
D-Glucose	0.05
D-Fructose	1.5

D-Glucose has a lower Km → greater affinity for hexokinase → hexokinase preferentially phosphorylates glucose over fructose.

Additional Km examples:

Enzyme	Substrate	Km (µM)
Chymotrypsin	Acetyl-L-tryptophanamide	5,000
Lysozyme	Hexa-N-acetylglucosamine	6
β-Galactosidase	Lactose	4,000
Carbonic anhydrase	CO₂	8,000
Arginine-tRNA synthetase	Arginine	3

7.5 Clinical and Experimental Uses of Km

Characterizing the number and types of substrates for a given enzyme.
Comparing similar enzymes from different tissues or organisms.
The Km of rate-limiting enzymes in metabolic pathways governs the amount of product and overall pathway regulation.
Clinically: Km comparisons help evaluate the effects of mutations on protein function in inherited diseases.

7.6 Multi-Substrate Reactions

Nearly two-thirds of all enzymatic reactions involve two substrates and two products (bi-substrate reactions). Example:

Hexokinase:
ATP + Glucose → ADP + Glucose-6-phosphate

Each substrate has its own Km value with the enzyme.

8. Factors Affecting Enzyme Activity

8.1 Substrate Concentration

Already covered in kinetics (Section 7).

8.2 Temperature

Reaction rate increases with temperature up to an optimum (typically ~37°C for human enzymes).
Above the optimum, denaturation occurs — the enzyme loses its three-dimensional structure and activity drops sharply.
This is why high fevers are dangerous and why storage of enzymes requires cold temperatures.

8.3 pH

pH affects enzyme activity in two ways:

On the enzyme:

Enzymes have ionizable groups on their active sites (e.g., His, Asp, Glu, Lys).
Changing pH alters the ionization state of these groups, disrupting the active site.
pH changes can also alter the overall 3D structure of the enzyme.

On the substrate:

Some substrates contain ionizable groups; pH affects their charge and thus their affinity for the enzyme.

Each enzyme has an optimum pH at which activity is maximal:

Enzyme	Optimum pH
Pepsin	1.5
Cholinesterase	7.0
Catalase	7.0
Fumarase	7.4
Ribonuclease	7.5
Arginase	9.7
Trypsin	~8.0

8.4 Enzyme Concentration [E]

At fixed, saturating [S], the initial rate is directly proportional to enzyme concentration.
This relationship holds as long as sufficient substrate is present.
This principle underlies gene induction and downregulation as mechanisms of metabolic control: increasing [E] speeds up a reaction; decreasing [E] slows it.

9. Enzyme Inhibition

Enzyme inhibitors are agents (metabolites, substrate analogs, toxins, drugs, metal complexes) that decrease enzyme activity. This is one of the fundamental mechanisms for biological control and pharmacological intervention.

Overview of Inhibition Types

Inhibition
├── Irreversible (covalent interactions)
└── Reversible (non-covalent/weak interactions)
    ├── Competitive
    └── Non-competitive (and Uncompetitive)

9.1 Reversible Inhibition

Competitive Inhibition

Mechanism:

The inhibitor has a structure similar to the substrate and binds to the same active site.
Enzyme cannot distinguish between substrate and inhibitor.
When inhibitor binds, substrate binding is prevented (they compete).
Inhibitor binding is reversible — increasing [S] displaces the inhibitor.

Effect on kinetics:

Vmax is unchanged (can still be achieved at very high [S]).
Km is increased (apparent Km, Km’) — more substrate needed to achieve half-maximal velocity.
As [I] increases, Km’ increases further.

Example:

Benzamidine competes with arginine for binding to trypsin (both have a positively charged guanidinium/amidinium group).
Allopurinol inhibits xanthine oxidase (used to treat gout): Hypoxanthine → Xanthine → Uric acid. Allopurinol competes with hypoxanthine/xanthine for the active site, reducing uric acid production.

Schematic:

E + S ⇌ ES → E + P
+
I
↕
EI (dead-end complex)

Non-Competitive Inhibition

Mechanism:

Inhibitor binds to a site different from the active site (an allosteric site).
Inhibitor and substrate can bind the enzyme simultaneously (EIS complex forms).
Increasing [S] cannot overcome inhibition, because the inhibitor doesn’t block substrate binding.

Effect on kinetics:

Vmax is decreased (fewer productive ES complexes even at saturation).
Km is unchanged (substrate can still bind with the same affinity).

Schematic:

E + S ⇌ ES → E + P
+ +
I  I
↕  ↕
EI + S ⇌ EIS (nonproductive)

Uncompetitive Inhibition

Inhibitor binds only to the ES complex, not free enzyme.
Both Vmax and Km are decreased by the same factor.
Produces parallel lines on a Lineweaver-Burk double-reciprocal plot.

Summary Table: Reversible Inhibition

Parameter	Competitive	Non-competitive	Uncompetitive
Vmax	Unchanged	Decreased	Decreased
Km	Increased	Unchanged	Decreased
Overcome by ↑[S]?	Yes	No	No
Binding site	Active site	Allosteric site	ES complex

9.2 Irreversible Inhibition

Irreversible inhibitors covalently modify an essential amino acid required for enzyme activity. The enzyme is permanently inactivated.

Types:

1. Group-Specific Reagents React with specific amino acid side chains.

Example: DIPF (diisopropylphosphofluoridate) — an organophosphate (related to nerve agents and pesticides) — reacts with the serine hydroxyl group in the active site of acetylcholinesterase, permanently inactivating it.

2. Substrate Analogs Structurally similar to the substrate; bind to the active site and covalently modify active-site residues.

Example: TPCK (Tosyl-L-phenylalanine chloromethyl ketone) — resembles chymotrypsin’s natural substrate and irreversibly alkylates His-57 in the active site.

3. Suicide Inhibitors (Mechanism-Based Inhibitors)

Relatively unreactive until they bind to the active site of a specific enzyme.
The enzyme initiates catalysis on the inhibitor, which is then converted into a highly reactive intermediate that covalently and irreversibly binds the enzyme.
Called “suicide” because the enzyme participates in its own irreversible inactivation.

Classic example — Penicillin:

Glycopeptide transpeptidase normally catalyzes the formation of cross-links in bacterial cell walls (between D-amino acids in peptidoglycan).
Penicillin mimics the D-Ala–D-Ala terminus of the peptidoglycan substrate.
The enzyme initiates a reaction with penicillin, forming a stable penicilloyl-enzyme complex via a serine residue — the enzyme is permanently inactivated.
Without functional transpeptidase, bacteria cannot build their cell walls → bacterial death.
Particularly effective against Gram-positive bacteria.

Bacterial resistance — β-Lactamase:

Some bacteria produce β-lactamase, an enzyme that hydrolyzes the β-lactam ring of penicillin before it can reach transpeptidase.
Clavulanic acid is a suicide inhibitor of β-lactamase — it binds irreversibly to β-lactamase, protecting penicillin. This is the basis of combination antibiotics (e.g., Augmentin = amoxicillin + clavulanic acid).

10. Regulation of Enzyme Activity

Cells must regulate enzyme activity to meet metabolic demands. There are several key mechanisms:

10.1 Substrate Concentration

As covered in Michaelis–Menten kinetics — the simplest form of regulation.

10.2 Regulation of Enzyme Concentration

The amount of enzyme in a cell can be changed by:

Induction (gene expression):

Hormones or growth factors trigger increased transcription and translation → more enzyme molecules are produced.
This is slow control (takes hours).

Degradation:

Lysosomes contain hydrolytic enzymes and degrade proteins non-specifically.
Proteasomes degrade proteins in a regulated, specific manner. The pathway:
1. Ubiquitin (a 76-amino-acid polypeptide) is covalently attached to the target protein.
2. The ubiquitinated protein is recognized and degraded by the 26S proteasome.
3. This is an irreversible process.

10.3 Reversible Covalent Modification — Phosphorylation

The most common regulatory covalent modification:

A phosphate group is transferred from ATP to a hydroxyl group on Ser, Thr, or Tyr residues.
Catalyzed by protein kinases.
Reversed by protein phosphatases (hydrolysis of the phosphoester bond).
Phosphorylation typically causes a conformational change that either activates or inactivates the enzyme.

Example — Glycogen Phosphorylase:

In its dephosphorylated form → inactive (phosphorylase b).
Phosphorylation by phosphorylase kinase → active (phosphorylase a).
This is a critical regulatory switch in glycogen metabolism.

Other covalent modifications include:

Adenylation
Uridylylation
ADP-ribosylation
Methylation
Myristoylation, palmitoylation, prenylation (lipid modifications that anchor proteins to membranes)

10.4 Allosteric Regulation

Definition: Allosteric comes from Greek: allos = “other,” stereos = “shape/site” — meaning regulation at “another site.”

An allosteric protein is one in which binding of a ligand at one site affects the binding properties of another site on the same protein.

Key features of allosteric enzymes:

Typically larger and more complex than non-allosteric enzymes, with two or more subunits.
Have allosteric sites distinct from the active site where effectors (modulators) bind.
Effectors can be positive (activators) or negative (inhibitors).
Do NOT obey Michaelis–Menten kinetics — instead show a sigmoidal (S-shaped) V₀ vs [S] curve due to cooperativity between subunits.
Regulatory enzymes in metabolic pathways are typically allosteric.

Cooperativity:

Binding of one substrate or effector molecule to one subunit induces conformational changes in other subunits, making it easier (positive cooperativity) or harder (negative cooperativity) for subsequent binding events.
Analogous to the oxygen-binding behavior of hemoglobin vs. myoglobin (hemoglobin is a classic allosteric protein).

Kinetic effects of allosteric modulators:

Allosteric activator (+): Shifts the sigmoidal curve toward a more hyperbolic profile (lower apparent Km, enzyme more responsive to substrate).
Allosteric inhibitor (−): Produces a more sigmoidal curve (higher apparent Km, enzyme less responsive to substrate).
At high [S], Vmax is still reached (the enzyme can still be saturated).

Types of allosteric regulation:

Homotropic (homo-allosteric): The normal substrate itself is the modulator — provides cooperative binding. Requires multimeric enzymes.
Heterotropic (hetero-allosteric): A molecule other than the substrate binds to the regulatory site. Provides feedback or feedforward regulation.

10.5 Feedback Inhibition and Feedforward Activation

Feedback inhibition: The end product of a metabolic pathway inhibits an enzyme early in the pathway, shutting down its own synthesis when sufficient product exists.

A →(E1)→ B →(E2)→ C →(E3)→ D →(E4)→ E →(E5)→ F (product)
         ↑__________________________|
                 Inhibition

The inhibited enzyme (E1) is typically allosteric.

Feedforward activation: A metabolite early in a pathway activates an enzyme further downstream, anticipating increased metabolic demand.

A →(E1)→ B →(E2)→ C →(E3)→ D →(E4)→ E →(E5)→ F
     └──────────────────────────────→ activates E4

Clinical relevance: Gout results from excess uric acid. Allopurinol inhibits xanthine oxidase (competitive inhibition) — a real-world example of using enzyme inhibition therapeutically.

11. Zymogens: Inactive Enzyme Precursors

Some enzymes are synthesized in an inactive form called a zymogen (or proenzyme). They are activated by proteolytic cleavage — removal of one or more peptide fragments — which causes a conformational change exposing the active site.

Why zymogens?

To prevent unwanted proteolytic damage in tissues where enzymes are synthesized (e.g., the pancreas).
Proteases of the digestive system and blood clotting cascade must be kept inactive until needed.

Digestive Enzyme Zymogens

Zymogen (pancreas)	Activating enzyme	Active enzyme
Trypsinogen	Enteropeptidase (intestine)	Trypsin
Chymotrypsinogen	Trypsin	Chymotrypsin + 2 dipeptides
Procarboxypeptidase	Trypsin	Carboxypeptidase

Chymotrypsin activation involves multiple cleavage steps of chymotrypsinogen, with key cuts at residues 13–14 and 15–16, and 147–148 and 149–150, producing the active enzyme held together by disulfide bonds.

Blood Clotting: A Zymogen Cascade

Blood clotting must occur rapidly after injury to prevent blood loss. This is achieved through a cascade of zymogen activations — each active enzyme activates the next zymogen in sequence, creating enormous signal amplification.

Two pathways converge:

Intrinsic pathway (triggered by damaged surfaces and contact factors)
Extrinsic pathway (triggered by tissue trauma releasing tissue factor)

Both converge on the Final Common Pathway:

Factor X is activated → Factor Xa
Factor Xa activates prothrombin → thrombin
Thrombin converts fibrinogen → fibrin (the clot)

Naming convention: Clotting factors are named by Roman numerals in order of discovery (not their order of action). The activated form adds the suffix “a” (e.g., Factor X → Factor Xa).

Clinical note: Deficiency in clotting factors causes hemophilia (e.g., Factor VIII deficiency = Hemophilia A). Warfarin inhibits vitamin K-dependent clotting factor synthesis, used as an anticoagulant.

Summary and Key Takeaways

Concept	Key Point
Ligand–protein binding	Specific, reversible, complementary; induced fit occurs
Enzymes	Protein catalysts; lower activation energy; highly specific
Active site	Complementary to transition state, not ground-state substrate
Binding energy	Weak interactions between enzyme and substrate; major energy source for catalysis
Lock & Key	Rigid model; historically important but insufficient
Induced Fit	Flexible active site; optimized for transition state
Km	[S] at ½Vmax; measure of affinity (lower = higher affinity)
Vmax	Maximum velocity at enzyme saturation
Competitive inhibition	Same active site; ↑Km, Vmax unchanged; overcome by ↑[S]
Non-competitive inhibition	Different site; ↓Vmax, Km unchanged; not overcome by ↑[S]
Irreversible inhibition	Covalent modification; permanent inactivation
Suicide inhibitors	Penicillin (transpeptidase), clavulanic acid (β-lactamase)
Allosteric regulation	Sigmoidal kinetics; effectors at non-active sites; cooperativity
Feedback inhibition	End product inhibits early pathway enzyme
Phosphorylation	Most common reversible covalent modification; kinases/phosphatases
Zymogens	Inactive precursors activated by proteolysis; protects tissues
Blood clotting cascade	Sequential zymogen activation; enormous amplification

Prepared from lecture slides — Alma Mater Studiorum, Università di Bologna
General Biochemistry — Enzymes and Enzymology

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