Enzyme Reaction Velocity Factors

• Enzyme activity can be increased or decreased by several factors

Factors affecting reaction velocity

• Factors affecting reaction velocity include substrate concentration, enzyme concentration, temperature, pH, and cofactors availability.

Substrate Concentration

• Substrate concentration indicates the amount of substrate available for the enzyme to act upon.
• At low substrate concentrations, reaction rate increases as substrate concentration increases.
• At high substrate concentrations, the enzyme becomes saturated when all active sites are occupied, leading to a plateau in reaction rate.

Enzyme concentration

• Enzyme concentration refers to the amount of enzyme available to catalyze the reaction.
• At sufficient substrate concentration, enzyme velocity increases as enzyme concentration increases.
• A higher enzyme concentration provides more active sites, enhancing enzyme-substrate interactions.
• Once the substrate is depleted or becomes limited, enzyme velocity decreases or stops.

Temperature

• Temperature is defined as the degree of heat energy in the environment.
• Reaction velocity increases as temperature rises, reaching a maximum at the enzyme's optimum temperature.
• Further temperature increase leads to a decline in reaction velocity due to enzyme denaturation, which is the irreversible loss of function due to unfolding of protein structure.
• Below the optimum temperature, reaction velocity decreases due to enzyme inactivation, which is reversible and caused by a lack of energy.
• Most human enzymes function optimally between 35°C and 40°C and begin to denature above 40°C.
• Thermophilic bacteria in hot springs have optimum temperatures around 70°C.

pH

• pH represents the acidity or alkalinity of the environment, measured on a scale from 0 to 14.
• Each enzyme has an optimum pH at which it achieves maximum velocity.
• At the optimum pH, the active site and substrate are in their ideal ionization state for effective interactions.
• Deviations above or below the optimum pH alters the ionization state of both the enzyme and the substrate, reducing binding efficiency and decreasing reaction velocity.
• Extreme pH values cause denaturation, leading to irreversible enzyme inactivation.

Proteolytic cleavage

• Some enzymes are synthesized as inactive proenzymes or zymogens, which become active only after being cleaved at a specific site in their polypeptide chain by specific proteases.
• Many digestive enzymes that hydrolyze proteins, such as trypsin and pepsin, are synthesized as zymogens in the stomach and pancreas.

Enzyme Inhibitors Definition

• Enzyme inhibitors are molecules that reduce or slow down enzymatic reactions.
• They are typically specific to certain enzymes and function effectively at low concentrations without destroying the enzyme.

Enzyme Inhibitors Importance

• Enzyme inhibitors regulate metabolic pathways by controlling enzyme activity.
• Enzyme inhibitors can be used as drugs in medical treatments.
• Enzyme inhibitors can be beneficial (as in drugs) or harmful (as in poisons).

Enzyme Inhibitors Examples

• Statins inhibit HMG-CoA reductase, lowering cholesterol levels.
• Aspirin irreversibly inhibits COX enzymes, reducing inflammation.
• Nerve toxins and poisons target enzymes in the nervous system, leading to toxicity.

Types of Inhibitors: Competitive Inhibition

• These molecules have a similar shape to the substrate and attach to the enzyme's active site, blocking the real substrate.
• Competitive inhibitors "compete" with the substrate, but adding more substrate can reduce their effect.
• Examples of competitive inhibitors include methotrexate, which inhibits dihydrofolate reductase and is used in chemotherapy, and sulfa drugs, which inhibit folic acid synthesis in bacteria.

Types of Inhibitors: Non-Competitive Inhibition

• Non-competitive inhibitors bind to an allosteric site, not the active site, and reduce enzyme function regardless of substrate concentration.
• Lead (Pb2+) poisoning affects multiple enzymes by binding allosterically.

Types of Inhibitors: Irreversible Inhibition

• Irreversible inhibitors cause permanent enzyme inactivation.
• Irreversible inhibitors permanently inactivate the enzyme through covalent binding to a certain group in the active site.
• Aspirin permanently inhibits COX enzymes, reducing inflammation.
• Nerve gases (organophosphates) irreversibly inhibit acetylcholinesterase, leading to paralysis.

Medical Relevance of Enzyme Inhibitors

• Many naturally occurring and synthetic compounds act as enzyme inhibitors.
• Enzyme inhibitors play a crucial role in therapeutic drug design, targeting specific enzymes to treat diseases.

Examples of Enzyme Inhibitors Used in Disease Treatment

• Antibacterials: Trimethoprim inhibits dihydrofolate reductase, and D-cycloserine inhibits alanine racemase
• Antifungals: Terbinafine inhibits fungal squalene epoxidase
• Antivirals: Cytosine arabinoside inhibits DNA, RNA polymerases, and acyclovir inhibits viral DNA polymerase
• Antiprotozoals: Alpha-difluoromethyl ornithine inhibits ornithine decarboxylase

Enzyme Inhibition in Poisoning Treatment

• Enzyme inhibitors are not always harmful; some are used to treat poisonings.
• Ethylene Glycol Poisoning (Car Antifreeze Poisoning)
  • Ethylene glycol is harmless initially but is converted into a toxic substance, oxalic acid (a deadly poison), by alcohol dehydrogenase. - Treatment: Ethanol (alcohol) is given as a competitive inhibitor and will compete with ethylene glycol for the active site of alcohol dehydrogenase, preventing ethylene glycol metabolism. - Result: Ethylene glycol is safely excreted from the body.

Enzymes in Clinical Diagnosis

• Plasma enzymes are divided into two main groups: specific and non-specific enzymes.

Specific Enzymes

• Specific enzymes are actively released into the blood by certain cells for specific functions, such as liver enzymes that help with blood clotting.

Non-Specific Enzymes

• Non-specific enzymes are released from cells during normal cell turnover.
• These enzymes usually work inside cells and have no specific role in the blood.
• Increased levels of non-specific enzymes in plasma may indicate tissue damage.
• In healthy individuals, the levels of these enzymes remain fairly stable.

How Are Enzymes Used in Diagnosis?

• Many diseases cause tissue damage, leading to the release of intracellular enzymes into the plasma.
• Measuring enzyme activity in plasma helps diagnose diseases affecting the heart, liver, skeletal muscles, and other tissues, as each organ has its specific enzymes, making diagnosis precise.
• The extent of enzyme elevation often correlates with the severity of tissue damage, making enzyme tests valuable for prognosis evaluation.
• Elevated cardiac enzymes indicate a heart attack (myocardial infarction). Higher enzyme levels indicate greater tissue damage

Isoenzymes & Their Role in Diagnosis

• Isoenzymes (Isozymes) are different molecular forms of the same enzyme that exist in various tissues.

Characteristics of Isoenzymes

• Catalyze the same reaction.
• Have different structures.
• Have different polypeptide chains, affecting their function.
• Different substrate affinities & response to activators/inhibitors.
• Help distinguish which organ is affected based on enzyme patterns.
• Lactate Dehydrogenase (LDH) Isoenzymes
  • LDH-1: Heart & RBCs - Elevated in myocardial infarction.
  • LDH-2: White blood cells
  • LDH-3: Lungs - Elevated in pulmonary diseases.
  • LDH-4 & LDH-5: Liver & Skeletal Muscle - Elevated in hepatitis & muscle injuries.

Liver Function Enzymes & Disease Diagnosis

• ALT (Alanine Aminotransferase)Indicates liver cell damage.
• AST (Aspartate Aminotransferase) Found in liver & muscle; high levels suggest severe damage.
• ALP (Alkaline Phosphatase) & GGT (Gamma-Glutamyl Transferase)Indicate bile duct obstruction or liver disease.
• Clinical Significance:
  • High ALT & AST Liver damage (e.g., hepatitis, alcohol abuse).
  • High ALP & GGT Biliary disease or bone disorders.

Cardiac Enzymes in Heart Attack Diagnosis

• When a heart attack occurs, cardiac muscle cells release enzymes into the blood. Key markers:
  • Troponins (cTnI, cTnT): Most specific for MI, peak at 24 hours.
  • CK-MB (Creatine Kinase-MB): Peaks 12-24 hours after MI, returns to normal faster than troponins.
  • LDH (Lactate Dehydrogenase): Older biomarker, less specific.

Pancreatic Enzymes for Diagnosing Pancreatitis

• Amylase & Lipase are key markers for acute pancreatitis.
• Elevated levels indicate pancreatic inflammation or obstruction. Comparison:
  • Amylase: Rises quickly, but less specific.
  • Lipase: More specific for pancreatic disorders.

Enzymes used as drugs

• Trypsin, lipase and amylase - Use for pancreatic insufficiency
• Alpha-1 Antitrypsin- Use for emphysema
• Chymotrypsin- Use for pain killer and anti-inflammatory

Enzymes used as tumor markers

• Serum acid phosphatase- Use for Prostate cancer
• B- Glucuronidase- Use for Cancer of urinary bladder

Enzymes used as reagents

• Uricase- Use to test Uric acid
• Glucose oxidase- Use to test Glucose