Enzyme Activity and Factors

• Enzyme activity can be increased or decreased by substrate concentration, enzyme concentration, temperature, pH, and cofactor availability.

Substrate Concentration

• Enzyme activity increases with substrate concentration until all active sites are occupied.
• When all active sites are occupied, the enzyme is considered saturated, and the reaction rate plateaus.
• At low substrate concentrations an increase in substrate concentration increases the reaction rate.

Enzyme Concentration

• Enzyme velocity increases as enzyme concentration increases, given sufficient substrate.
• More enzymes lead to more available active sites, which enhances enzyme-substrate interactions to speed up the reaction.
• If the substrate is limited the enzyme velocity will decrease or stop.

Temperature

• Reaction velocity increases with temperature until it reaches the enzyme's optimum temperature.
• Further temperature increases above its optimum temperature will lead to enzyme denaturation and a decline in reaction velocity.
• Denaturation involves the unfolding of the protein structure, which is irreversible.
• Below the optimum temperature, the reaction velocity decreases due to enzyme inactivation, which is reversible due to a lack of energy.
• Most human enzymes function optimally between 35°C and 40°C, but begin to denature above 40°C.
• Thermophilic bacteria have optimum temperatures around 70°C.

pH

• Each enzyme has an optimal pH at which it achieves maximum velocity.
• The active site and substrate are in their ideal ionization state for effective interactions at the optimal pH.
• Deviations above or below the optimum pH reduce binding efficiency and decrease reaction velocity due to altered ionization states of the enzyme and substrate
• Extreme pH values cause denaturation, leading to irreversible enzyme inactivation.

Proteolytic Cleavage

• Enzymes can be synthesized as inactive proenzymes or zymogens and become active only after being cleaved at a specific site by specific proteases.
• Digestive enzymes like 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, are specific to certain enzymes and function effectively at low concentrations without destroying the enzyme.
• They regulate metabolic pathways by controlling enzyme activity.
• Enzyme inhibitors can be used as drugs in medical treatments, or they can be harmful as poisons.
  • 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.

Competitive Inhibition

• Competitive inhibitors have a similar shape to the substrate and attach to the enzyme's active site.
• Competitive inhibitors block the real substrate and "compete" with it.
• Adding more substrate can reduce the effect of the inhibitor.
  • Methotrexate inhibits dihydrofolate reductase and is used in chemotherapy.
  • Sulfa drugs inhibit folic acid synthesis in bacteria.

Non-Competitive Inhibition

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

Irreversible Inhibition

• Irreversible inhibitors result in permanent enzyme inactivation.
• The mechanism is by covalent binding or a certain group in the active site.
  • Aspirin permanently inhibits COX enzymes, which reduces inflammation.
  • Nerve gases (organophosphates) irreversibly inhibit acetylcholinesterase, leading to paralysis.

Medical Relevance of Enzyme Inhibitors

• Naturally occurring and synthetic compounds act as enzyme inhibitors, they are therapeutically useful by targeting specific enzymes to treat diseases.
  • Trimethoprim inhibits Dihydrofolate reductase as an antibacterial.
  • D-cycloserine inhibits Alanine racemase as an antibacterial.
  • Terbinafine inhibits Fungal squalene epoxidase as an antifungal.
  • Cytosine arabinoside inhibits DNA, RNA polymerases as an antiviral.
  • Acyclovir inhibits Viral DNA polymerase as an antiviral.
  • Alpha-difluoromethyl ornithine inhibits Ornithine decarboxylase as an antiprotozoal.

Enzyme Inhibition in Poisoning Treatment

• Enzyme inhibitors are not always harmful, some are used to treat poisonings.
  • Ethylene glycol is harmless initially, but alcohol dehydrogenase converts it into toxic oxalic acid. This causes Ethylene Glycol Poisoning.
  • Treatment involves administering ethanol, a competitive inhibitor, to compete with ethylene glycol for the active site of alcohol dehydrogenase, preventing ethylene glycol metabolism.
  • Ultimately, ethylene glycol is safely excreted from the body.

Enzymes in Clinical Diagnosis

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

Specific Enzymes

• Actively released into the blood by certain cells for specific functions.
• Liver secretes specific enzymes that help with blood clotting.

Non-Specific Enzymes

• A larger group of enzymes released from cells during normal cell turnover.
• Enzymes usually have no specific role in the blood and work inside cells.
• Elevated presence of these enzymes in plasma may indicate tissue damage.
• Levels of these enzymes remain stable in healthy individuals.

How Are Enzymes Used in Diagnosis?

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

Isoenzymes & Their Role in Diagnosis

• Isoenzymes or isozymes are different molecular forms of the same enzyme that exist in various tissues.
• Characteristics of isoenzymes:
  • Catalyze the same reaction
  • Different structures
  • Different polypeptide chains, affecting their function
  • Different substrate affinities and varying responses to activators/inhibitors
  • Help distinguish which organ is affected based on enzyme patterns.

Lactate Dehydrogenase (LDH) Isoenzymes

• LDH-1 is found in the Heart & RBCs and is elevated in myocardial infarction.
• LDH-2 is found in white blood cells.
• LDH-3 is found in the lungs and is elevated in pulmonary diseases.
• LDH-4 & LDH-5 are found in the Liver & Skeletal Muscle and is elevated during hepatitis & muscle injuries.

Liver Function Enzymes & Disease Diagnosis

• Key Liver Enzymes:
  • ALT (Alanine Aminotransferase) indicates liver cell damage.
  • AST (Aspartate Aminotransferase) is found in the 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 levels indicate liver damage.
  • High ALP & GGT levels indicate biliary disease or bone disorders.

Cardiac Enzymes in Heart Attack Diagnosis

• Cardiac muscle cells release enzymes into the blood when a heart attack occurs.
  • Troponins (cTnI, cTnT) are the most specific markers for myocardial infarction, peaking at 24 hours.
  • CK-MB (Creatine Kinase-MB) peaks 12-24 hours after myocardial infarction; returns to normal faster than troponins.
  • LDH (Lactate Dehydrogenase) is an older, less specific biomarker.

Pancreatic Enzymes for Diagnosing Pancreatitis

• Amylase & Lipase are key markers for acute pancreatitis.
• Elevated levels indicate pancreatic inflammation or obstruction.
• Amylase rises quickly, but is less specific to the pancreas.
• Lipase is more specific for pancreatic disorders.

Enzymes Used as Drugs

• Trypsin, lipase, and amylase together are used for pancreatic insufficiency.
• Alpha-1 Antitrypsin is used for Emphysema.
• Chymotrypsin is used as a painkiller and anti-inflammatory.

Enzymes Used as Tumor Markers

• Serum acid phosphatase indicates prostate cancer.
• B-Glucuronidase indicates cancer of the urinary bladder.

Enzymes Used as Reagents

• Uricase is used to test for Uric acid.
• Glucose oxidase is used to test for Glucose.