ENZYMES AND BIOCHEMICAL REACTIONS

 1. Introduction to Enzymes

Enzymes are biological catalysts that accelerate biochemical reactions without being consumed during the reaction. Most enzymes are proteins, although some RNA molecules, known as ribozymes, also possess catalytic activity.
Enzymes are essential for almost all metabolic processes, including digestion, energy production, protein synthesis, and cellular regulation.

Major characteristics of enzymes:

1. They increase the rate of biochemical reactions.
2. They lower the activation energy required for a reaction.
3. They are not consumed during the reaction.
4. They exhibit substrate specificity.
5. They function optimally under specific temperature and pH conditions.
6. Many enzyme-catalysed reactions are reversible.

1.1 Enzyme Nomenclature

Enzymes are commonly named by adding the suffix -ase to the substrate or type of reaction they catalyse.

Examples:

• Amylase: breaks down starch.
• Lipase: breaks down lipids.
• Protease: breaks down proteins.
• Lactase: breaks down lactose.
• Urease: catalyses the breakdown of urea.

1.2 Classification of Enzymes

According to the International Union of Biochemistry and Molecular Biology (IUBMB), enzymes are classified into six major traditional classes, with a seventh class added in the modern EC classification.

Enzyme class	Function	Example
Oxidoreductases	Catalyse oxidation-reduction reactions	Lactate dehydrogenase
Transferases	Transfer functional groups between molecules	Aminotransferases
Hydrolases	Break chemical bonds using water	Lipase, amylase
Lyases	Remove or add groups without hydrolysis or oxidation	Aldolase
Isomerases	Convert molecules into their isomers	Phosphoglucose isomerase
Ligases	Join two molecules using energy, usually ATP	DNA ligase
Translocases	Catalyse movement of ions or molecules across membranes	ATP synthase (translocation-related activity)

2. Enzyme Structure and Function

2.1 Structure of Enzymes

Most enzymes are globular proteins with specific three-dimensional structures. Their structure determines their catalytic activity and substrate specificity.

2.2 Active Site

The active site is the specific region of an enzyme where the substrate binds and the catalytic reaction takes place.
Important features:

• It has a specific three-dimensional shape.
• It contains amino acid residues involved in substrate binding and catalysis.
• It provides a suitable environment for the biochemical reaction.
• It determines much of the enzyme's substrate specificity.

2.3 Models of Enzyme Action

A. Lock-and-Key Model

Proposed by Emil Fischer, this model suggests that the active site has a fixed shape complementary to the substrate. The substrate fits into the active site like a key fits into a lock.

B. Induced-Fit Model

Proposed by Daniel Koshland, this model suggests that the active site changes its shape slightly when the substrate binds. This conformational change improves substrate binding and facilitates catalysis.

2.4 Mechanism of Enzyme Action

Enzymes accelerate reactions by lowering the activation energy.
The general steps are:

1. The substrate approaches the enzyme's active site.
2. The substrate binds to the active site.
3. An enzyme-substrate complex forms.
4. The enzyme facilitates the conversion of substrate into product.
5. The product is released.
6. The enzyme remains available for another reaction.

Activation energy: The minimum energy required for reactants to reach the transition state and undergo a chemical reaction.

3. Cofactors and Coenzymes

Some enzymes require additional non-protein components to perform their catalytic functions. These components are called cofactors.

3.1 Cofactors

A cofactor is a non-protein substance required for the activity of certain enzymes.
Cofactors are broadly classified into:
A. Inorganic cofactors
These are usually metal ions required for enzyme activity.
Examples:

Metal ion	Enzyme example
Magnesium (Mg²⁺)	Kinases
Zinc (Zn²⁺)	Carbonic anhydrase
Iron (Fe²⁺/Fe³⁺)	Catalase and peroxidase
Copper (Cu²⁺)	Cytochrome c oxidase
Manganese (Mn²⁺)	Superoxide dismutase

B. Organic cofactors

These are organic molecules that assist enzyme activity. They are commonly known as coenzymes.

3.2 Coenzymes

Coenzymes are small organic molecules that assist enzymes in transferring chemical groups, electrons, or atoms during biochemical reactions.
Most coenzymes are derived from vitamins, particularly B-complex vitamins.

Coenzyme	Vitamin source	Major function
NAD⁺	Niacin (B3)	Transfers electrons in oxidation-reduction reactions
NADP⁺	Niacin (B3)	Participates in biosynthetic reactions
FAD	Riboflavin (B2)	Transfers electrons and hydrogen
FMN	Riboflavin (B2)	Participates in electron transport
Coenzyme A	Pantothenic acid (B5)	Transfers acyl groups
TPP	Thiamine (B1)	Facilitates oxidative decarboxylation
PLP	Pyridoxine (B6)	Participates in amino acid metabolism
Biotin	Biotin (B7)	Transfers carbon dioxide in carboxylation reactions

3.3 Apoenzyme, Holoenzyme and Prosthetic Group

• Apoenzyme: The protein portion of an enzyme without its required cofactor; generally inactive.
• Cofactor: The non-protein component required for enzyme activity.
• Holoenzyme: The complete, catalytically active enzyme consisting of an apoenzyme and its required cofactor.
• Prosthetic group: A cofactor that is tightly or permanently associated with an enzyme.

3.4 Importance of Vitamins in Enzyme Function

Many vitamins act as precursors of coenzymes. Vitamin deficiencies can therefore impair enzyme activity and disrupt metabolic pathways.

Examples:

• Vitamin B1 deficiency can impair carbohydrate metabolism.
• Vitamin B3 deficiency can affect NAD⁺ and NADP⁺-dependent reactions.
• Vitamin B6 deficiency can affect amino acid metabolism.
• Vitamin B12 deficiency can interfere with DNA synthesis and certain metabolic reactions.

4. Enzyme Kinetics and Regulation

Enzyme kinetics is the study of the rates of enzyme-catalysed reactions and the factors affecting these rates.

4.1 Factors Affecting Enzyme Activity

1. Enzyme concentration
When substrate is available in excess, increasing enzyme concentration increases the reaction rate because more active sites are available.
2. Substrate concentration
Increasing substrate concentration generally increases the reaction rate until the enzyme approaches saturation.
3. Temperature
Enzyme activity generally increases with temperature up to an optimum level. Above this temperature, enzyme structure may be disrupted, resulting in reduced activity.
4. pH
Each enzyme has an optimum pH at which it shows maximum activity.
Examples:

Enzyme	Approximate optimum pH
Pepsin	1.5–2.0
Salivary amylase	6.7–7.0
Trypsin	7.5–8.5
Alkaline phosphatase	9–10

5. Inhibitors
Certain substances decrease enzyme activity by interfering with substrate binding or catalytic activity.

4.2 Michaelis-Menten Kinetics

Michaelis-Menten kinetics describes the relationship between substrate concentration and the rate of an enzyme-catalysed reaction. As substrate concentration increases, reaction velocity increases until the enzyme becomes saturated and reaches its maximum velocity (Vmax).

The Michaelis-Menten equation is:

Where Km is the substrate concentration at which the reaction velocity is half of Vmax.

4.3 Enzyme Inhibition

Enzyme inhibition occurs when a substance decreases or prevents enzyme activity.
The major types are:

A. Competitive inhibition

• The inhibitor competes with the substrate for the active site.
• It usually resembles the substrate structurally.
• Its effect can be overcome by increasing substrate concentration.

Example: Malonate inhibits succinate dehydrogenase by competing with succinate.

B. Non-competitive inhibition

• The inhibitor binds at a site other than the active site.
• It reduces catalytic activity.
• Increasing substrate concentration cannot fully overcome the inhibition.

C. Uncompetitive inhibition

• The inhibitor binds only to the enzyme-substrate complex

4.4 Regulation of Enzyme Activity

Enzyme regulation controls the rate of biochemical reactions according to the metabolic requirements of the cell.
Major mechanisms include:

1. Allosteric regulation: Regulatory molecules bind to a site other than the active site and change enzyme activity.
2. Feedback inhibition: The final product of a metabolic pathway inhibits an earlier enzyme in that pathway.
3. Covalent modification: Enzyme activity changes through chemical modification, such as phosphorylation or dephosphorylation.
4. Proteolytic activation: Some enzymes are produced as inactive precursors (zymogens) and activated by cleavage of specific peptide bonds.
5. Enzyme synthesis and degradation: Cells regulate enzyme concentration by controlling their production and breakdown.

Examples:

• Phosphofructokinase-1 (PFK-1) is regulated by ATP, AMP, and fructose-2,6-bisphosphate.
• Glycogen phosphorylase is regulated through phosphorylation and allosteric mechanisms.
• Trypsinogen is converted into active trypsin in the small intestine.

5. Clinical Significance of Enzymes

Enzymes are important diagnostic markers in clinical laboratory medicine. Damage to tissues and organs can cause intracellular enzymes to enter the bloodstream, resulting in increased serum enzyme activities. Measurement of enzyme activity helps in the diagnosis, assessment, and monitoring of several diseases.

5.1 Important Diagnostic Enzymes

5.2 Liver Enzymes

ALT (Alanine Aminotransferase)
ALT is an intracellular enzyme found predominantly in hepatocytes. Increased serum ALT activity is commonly associated with hepatocellular injury, such as viral hepatitis and drug-induced liver injury.
AST (Aspartate Aminotransferase)
AST is present in the liver, heart, skeletal muscles, and other tissues. Increased AST may occur in liver disease, muscle injury, and other conditions. It is less specific for liver injury than ALT.
ALP (Alkaline Phosphatase)
ALP is found mainly in the liver, bone, intestine, and placenta. Increased ALP may indicate cholestasis or increased bone activity.
GGT (Gamma-Glutamyl Transferase)
GGT is an enzyme associated with the hepatobiliary system. It is useful in evaluating the source of increased ALP. Concurrent elevation of ALP and GGT supports a hepatobiliary origin.

5.3 Cardiac and Muscle Enzymes

Creatine Kinase (CK)
CK catalyses the reversible transfer of a phosphate group between ATP and creatine phosphate. It is present mainly in skeletal muscle, cardiac muscle, and brain.
Its major isoenzymes include:

• CK-MM: Predominantly skeletal muscle.
• CK-MB: More abundant in cardiac muscle.
• CK-BB: Predominantly brain and other tissues.

CK-MB may increase following myocardial injury, but cardiac troponins are preferred biomarkers for diagnosing acute myocardial infarction.
Lactate Dehydrogenase (LDH)
LDH catalyses the reversible conversion of pyruvate into lactate. It is present in many tissues, including the liver, heart, skeletal muscle, and red blood cells. Increased LDH may indicate tissue damage, hemolysis, or other pathological conditions. It is not specific to a particular organ.

5.4 Pancreatic Enzymes

Amylase
Amylase breaks down starch into smaller carbohydrate molecules. Increased serum amylase may occur in acute pancreatitis, salivary gland disorders, and other conditions.
Lipase
Lipase catalyses the breakdown of triglycerides into fatty acids and glycerol. Serum lipase is generally more specific for acute pancreatitis than amylase.