Enzyme Reaction Rate Factors

• Enzymes are influenced by factors that either increase or decrease the reaction rate.
• These factors include substrate concentration, enzyme concentration, temperature, pH, and cofactors availability.

Substrate Concentration

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

Enzyme Concentration

• Enzyme concentration refers to the amount of enzyme available to catalyze a reaction.
• With a sufficient substrate concentration, enzyme velocity increases as enzyme concentration increases.
• Increased enzyme concentration provides more active sites, enhancing enzyme-substrate interactions, and accelerates reaction rate.
• Enzyme velocity decreases/stops when substrate is depleted or becomes limited.

Temperature

• Temperature can be defined as the degree of heat energy in the environment.
• Reaction velocity increases as temperature rises until it reaches a max at the enzyme's optimum temperature.
• Increased temperature above the optimum levels leads to a decline in reaction velocity, which results in enzyme denaturation, with loss of function due to the unfolding of protein structures (irreversible).
• Reaction velocity decreases due to enzyme inactivation (lack of energy), which is reversible when below optimal temperature.
• Most human enzymes function optimally between 35°C and 40°C, but begin to denature above 40°C.
• Some thermophilic bacteria in hot springs have optimum temperatures around 70°C.

pH

• pH is the acidity or alkalinity of the environment measured on a scale from 0-14.
• Each enzyme has an optimal pH to achieve maximum velocity.
• At optimal pH, the active site and substrate are in ideal ionization state, allowing effective interactions.
• pH deviations above or below the optimum alter 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

• Certain enzymes are synthesized as proenzymes or zymogens, which are inactive forms of enzymes that 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

• Enzyme inhibitors are molecules that reduce or slow down enzymatic reactions.
• Enzyme inhibitors are typically specific to certain enzymes and function effectively at low concentrations without destroying the enzyme.
• Enzyme inhibitors regulate metabolic pathways by controlling enzyme activity.
• Enzyme inhibitors can be used as drugs for medical treatments.
• Enzyme inhibitors can be both beneficial (drugs) or harmful (poisons).
• Examples:
  • Statins inhibit HMG-CoA reductase, lowering cholesterol levels.
  • Aspirin irreversibly inhibits COX enzymes, reducing inflammation.
  • Nerve toxins/poisons target enzymes in the nervous system, leading to toxicity.

Competitive Inhibition

• Competitive inhibitors 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 (higher concentration wins!).
• Competitive inhibition examples:
  • Methotrexate inhibits dihydrofolate reductase, used in chemotherapy.
  • Sulfa drugs inhibit folic acid synthesis in bacteria.

Non-Competitive Inhibition

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

Irreversible Inhibition

• Irreversible Inhibitors undergo permanent enzyme inactivation.
• Irreversible Inhibitors permanently inactivate the enzyme by covalent binding to a certain group in the active site.
• Example:
  • Aspirin permanently inhibits COX enzymes (reduces 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 include:
  • Antibacterial that inhibits Dihydrofolate reductase: Trimethoprim
  • Antibacterial that inhibits Alanine racemase: D-cycloserine
  • Antifungal that inhibits Fungal squalene epoxidase: Terbinafine
  • Antiviral that inhibits DNA, RNA polymerase: Cytosine arabinoside
  • Antiviral that inhibits Viral DNA polymerase: Acyclovir
  • Antiprotozoal that inhibits Ornithine decarboxylase: Alpha-difluoromethyl ornithine

Enzyme Inhibition in Poisoning Treatment

• Enzyme inhibitors are not always harmful and can be used to treat poisonings.
• Example:
  • 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.
      • To treat poisoning as a result of alcohol dehydrogenase, ethanol (alcohol) is given as a competitive inhibitor.
      • Ethanol competes with ethylene glycol for the active site of alcohol dehydrogenase.
      • This helps prevent Ethylene glycol metabolism and allows it to safely leave the body.

Enzymes in Clinical Diagnosis

• Plasma enzymes are divided into two main groups:
  • Specific Enzymes:
  • A small group of enzymes actively released into the blood by certain cells for specific functions.
  • The liver secretes specific enzymes for blood clotting.
  • Non-Specific Enzymes:
  • A larger group of enzymes released from cells during normal cell turnover.
  • The enzymes usually work inside cells and have no specific role in the blood.
  • If the levels of nonspecfic enzymes in plasma increase, it may indicate tissue damage.
  • Enzyme levels remain fairly stable in healthy individuals.

How are Enzymes Used in Diagnosis?

• Many diseases can 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, because 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 can indicate a heart attack (myocardial infarction).
  • Higher enzyme levels indicates greater tissue damage → which helps in prognosis evaluation.

Isoenzymes & Their Role in Diagnosis

• Isoenzymes (Isozymes) are different molecular forms of the same enzyme that exist in various tissues.
• Isoenzyme Characteristics:
  • Catalyze the same reaction.
  • Have different structures.
  • Have different polypeptide chains, affecting their function.
  • Have different substrate affinities and responses to activators/inhibitors.
  • Help distinguish which organ is affected depending on different enzyme patterns.
• Lactate Dehydrogenase (LDH) Isoenzymes:
  • LDH-1 can be found in the Heart and RBCs. Elevated in myocardial infarction.
  • LDH-2 can be found in white blood cells.
  • LDH-3 can be found in the Lungs and is elevated in pulmonary diseases.
  • LDH-4 & LDH-5 can be found in Liver & Skeletal Muscle is elevated hepatitis & muscle injuries.

Liver Function Enzymes & Disease Diagnosis

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

Cardiac Enzymes in Heart Attack Diagnosis

• Cardiac muscle cells release enzymes into the blood during a heart attack. Cardiac Enzyme Key markers:
  • Troponins (cTnI, cTnT) are most specific for MI, and peak at 24 hours.
  • CK-MB (Creatine Kinase-MB) peaks 12-24 hours after MI, and returns to normal faster than troponins.
  • LDH (Lactate Dehydrogenase) is an older biomarker, and is less specific.

Pancreatic Enzymes for Diagnosing Pancreatitis

• Key markers for acute pancreatitis are Amylase & Lipase.
• Elevated levels indicates pancreatic inflammation or obstruction.
• Comparison:
  • Amylase rises quickly, but is less specific.
  • Lipase is more specific for pancreatic disorders.

Enzymes used as drugs

• Trypsin, lipase, amylase is used for Pancreatic insufficiency.
• Alpha- 1 Antitrypsin is used for Emphysema.
• Chymotrypsin is used as a Pain killer and Anti-inflammatory.

Enzymes used as tumor markers

• Serum acid phosphatase is used for testing Prostate cancer.
• B- Glucuronidase test is used for testing Cancer of urinary bladder.

Enzymes used as reagents

• Uricase is used for testing Uric acid.
• Glucose oxidase is used for testing Glucose.