Section: Diagnostic Importance of Enzymes
Case Scenario 1:
A 58-year-old male is brought to the casualty at 4:00 AM with complaints of profuse sweating and a feeling of tightness in the chest for the past 3 hours. The chest pain radiates to his left shoulder and arm. ECG reveals ST-segment elevation and T-wave inversion.
- What is the most probable diagnosis?
- Which biochemical tests can help confirm this diagnosis?
- Describe the diagnostic enzymes, their pattern of change in concentration over time, and their significance.
- Add a brief note on the role of isoenzymes in the above condition.
Answer:
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- Most Probable Diagnosis:
Acute Myocardial Infarction (ST-Elevation Myocardial Infarction – STEMI)
- Biochemical Tests to Confirm Diagnosis:
- Cardiac Troponins (Troponin I, Troponin T)
- Creatine Kinase-MB (CK-MB)
- Lactate Dehydrogenase (LDH)
- Myoglobin (early but non-specific marker)
- Diagnostic Enzymes: Pattern of Change & Significance:
Marker Rises (hrs) Peaks (hrs) Returns to Normal (days) Significance Troponin I/T 3–6 12–24 7–10 Most specific & sensitive for MI CK-MB 3–6 18–24 2–3 Useful for reinfarction (due to short half-life) LDH 12–24 48–72 7–10 Late marker; not routinely used now Myoglobin 1–4 6–9 <24 hours Early rise, but not specific - Role of Isoenzymes in MI:
- CK has 3 isoenzymes: CK-MM (muscle), CK-BB (brain), CK-MB (heart-specific).
- CK-MB is more specific to cardiac tissue and helps differentiate cardiac vs. skeletal muscle damage.
- LDH has 5 isoenzymes; in MI, LDH-1 > LDH-2 (known as LDH flip), supporting the diagnosis in late presenters
Case Scenario 2:
A healthcare worker posted at a rural health centre develops loss of appetite, nausea, fever, and later develops jaundice. On investigation, he tests positive for Hepatitis B virus infection.
- Which enzymes are of diagnostic importance in this condition?
- Mention the class of enzyme to which these diagnostic enzymes belong.
- Classify enzymes, giving suitable examples from this case.
Answer:
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- a) Enzymes of Diagnostic Importance in Hepatitis B:
- Alanine aminotransferase (ALT / SGPT)
- Aspartate aminotransferase (AST / SGOT)
- Alkaline phosphatase (ALP)
- Gamma-glutamyl transferase (GGT)
- 5′-nucleotidase (optional, supports cholestasis diagnosis)
ALT is more specific to liver injury, while AST is present in both liver and other tissues (e.g., muscle).
- b) Class of Enzymes to Which These Belong:
- ALT and AST → Transferases
- ALP and GGT → Hydrolases
- c) Classification of Enzymes with Examples
Enzyme Class Function Example Oxidoreductases Catalyze oxidation–reduction reactions LDH, MDH Transferases Transfer functional groups between molecules ALT, AST Hydrolases Catalyze hydrolysis of bonds ALP, GGT Lyases Add or remove groups without hydrolysis Aldolase Isomerases Catalyze isomerization reactions Phosphohexose isomerase Ligases Join two molecules using ATP Glutamine synthetase
Case Scenario 3:
A 40-year-old male presents with severe epigastric pain radiating to the back, along with vomiting and abdominal distension. His serum shows markedly elevated levels of serum amylase and lipase enzymes.
- What is the most probable diagnosis?
- Mention the class of these enzymes and explain their diagnostic significance.
Answer:
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- Acute Pancreatitis
- Class of These Enzymes and Their Diagnostic Significance:
Enzyme Class Diagnostic Significance Amylase Hydrolase (specifically, an enzyme that breaks down starch into sugars) Early marker; rises within 6-12 hours of onset; elevated in pancreatitis but also in other conditions (e.g., salivary gland disorders). Lipase Hydrolase (breaks down triglycerides into fatty acids and glycerol) More specific for pancreatitis; rises within 4-8 hours, peaks at 24 hours, remains elevated longer than amylase. Helps confirm diagnosis. - Both enzymes are secreted by the pancreas and their elevated serum levels indicate pancreatic inflammation and injury.
- Lipase is preferred for diagnosis because of higher specificity and prolonged elevation.
Case Scenario 4:
A 55-year-old female presents with yellowish discoloration of skin and eyes, dark-colored urine, pale stools, and generalized itching. On investigation, she is found to have obstructive jaundice.
- Name the enzymes of diagnostic importance in this condition.
- Mention the class of these enzymes and explain their significance in the diagnosis of obstructive jaundice.
- How do the enzyme levels differ from those seen in hepatocellular jaundice?
Answer:
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- a) Enzymes of Diagnostic Importance in Obstructive Jaundice:
- Alkaline Phosphatase (ALP)
- Gamma-Glutamyl Transferase (GGT)
- 5′-Nucleotidase (optional, supports cholestasis diagnosis)
- b) Class of These Enzymes and Their Significance:
Enzyme Enzyme Class Diagnostic Significance Alkaline Phosphatase (ALP) Hydrolase Elevated in cholestasis due to increased synthesis and release from bile duct epithelium. Indicates bile duct obstruction. Gamma-Glutamyl Transferase (GGT) Transferase Elevated in obstructive jaundice; helps confirm hepatic origin of raised ALP (distinguishes from bone disease). 5′-Nucleotidase Hydrolase Another cholestatic enzyme elevated in obstruction. - Elevated ALP and GGT reflect cholestasis and biliary obstruction.
- These enzymes increase as a result of bile duct epithelial damage and bile flow blockage.
- c) Enzyme Levels in Obstructive Jaundice vs Hepatocellular Jaundice:
Enzyme/Marker Obstructive Jaundice Hepatocellular Jaundice ALP Markedly elevated Mild to moderate elevation GGT Markedly elevated Mild to moderate elevation AST (SGOT) & ALT (SGPT) Mild to moderate elevation Marked elevation (reflects hepatocyte injury) - In obstructive jaundice, cholestatic enzymes (ALP, GGT) rise more significantly due to bile duct obstruction.
- In hepatocellular jaundice, transaminases (AST, ALT) rise more prominently due to liver cell damage.
Case Scenario 5:
A 12-year-old boy presents with bone pain, bowing of legs, and delayed growth. On examination, he has widened wrists and prominent costochondral junctions. His biochemical investigations reveal elevated levels of a specific enzyme associated with bone turnover.
- What is the probable diagnosis?
- Name the enzyme of diagnostic importance in this condition.
- Mention the class of this enzyme and explain its role in the diagnosis of bone disorders.
Answer:
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- a) Probable Diagnosis:
Nutritional Rickets (most likely due to Vitamin D deficiency)
- b) Enzyme of Diagnostic Importance:
Alkaline Phosphatase (ALP)
- c) Enzyme Class and Diagnostic Role:
- Class: Hydrolase
(Specifically, catalyzes hydrolysis of phosphate esters) - Diagnostic Significance:
- ALP is produced by osteoblasts during bone formation.
- In rickets, bone mineralization is impaired, leading to increased osteoblastic activity.
- This results in elevated serum ALP, reflecting increased bone turnover and active bone remodeling.
- High ALP is a sensitive marker for metabolic bone diseases such as rickets, osteomalacia, and Paget’s disease.
Case Scenario 6:
Ravi, a 52-year-old male, visits the emergency department with complaints of severe chest pain radiating to his left arm, shortness of breath, and sweating. The physician suspects a myocardial infarction (heart attack) and orders blood tests, including measurement of creatine kinase (CK) and lactate dehydrogenase (LDH) levels. The results show an increase in CK-MB and LDH-1, while other isoenzymes remain within normal range. These findings support the diagnosis of acute myocardial infarction.
- Define isoenzymes and explain how they differ from regular enzymes.
- Describe the clinical importance of isoenzymes in diagnosis of myocardial infarction, with reference to CK and LDH isoenzymes.
- Name any two other enzymes or isoenzymes used in clinical diagnosis and state the condition(s) they help identify.
Answer:
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- a) Definition and Difference of Isoenzymes:
- Isoenzymes (isozymes) are different molecular forms of the same enzyme that catalyze the same chemical reaction but differ in amino acid sequence, structure, kinetic properties, and tissue distribution.
- Unlike regular enzymes (single form), isoenzymes help identify the tissue of origin due to their tissue-specific expression.
- b) Clinical Importance of Isoenzymes in Myocardial Infarction:
- Creatine Kinase (CK) has 3 isoenzymes:
- CK-MM → Skeletal muscle
- CK-BB → Brain
- CK-MB → Cardiac muscle (specific for myocardium)
- Lactate Dehydrogenase (LDH) has 5 isoenzymes:
- LDH-1 (Heart, RBCs)
- LDH-2 (Reticuloendothelial system)
- Normally, LDH-2 > LDH-1
- In MI, this ratio flips → LDH-1 > LDH-2 (LDH flip)
- In acute myocardial infarction (MI):
- Elevated CK-MB confirms cardiac muscle damage.
- Elevated LDH-1 further supports MI, especially in late presenters.
- c) Two Other Enzymes/Isoenzymes in Clinical Diagnosis:
Enzyme/Isoenzyme Condition Identified Alanine Aminotransferase (ALT/SGPT) Liver damage / Hepatitis Amylase or Lipase Acute pancreatitis
Subsection: Factors Affecting Enzyme Activity
Case Scenario 7:
Anita, an 18-year-old first-year college student, is performing a biology lab experiment to study the activity of the enzyme amylase, which breaks down starch into sugars. She notices that the rate of the reaction changes under different conditions.
In one trial, she performs the reaction at room temperature (25°C) and observes moderate enzyme activity. In another trial, she increases the temperature to 60°C and observes that the enzyme activity decreases significantly. In a third trial, she adjusts the pH of the solution from 7 to 3 and again notices a drop-in activity. She also observes that increasing the concentration of enzyme speeds up the reaction, but only up to a certain point, after which the rate does not increase further.
- Describe three factors that affect enzyme activity, using Anita’s experiment as a reference.
- Explain why enzyme activity decreased at 60°C and at pH 3 in Anita’s experiment.
Answer:
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- a) Three Factors Affecting Enzyme Activity (with Reference to Anita’s Experiment):
- Temperature:
- Moderate activity at 25°C (room temperature).
- Decreased activity at 60°C due to enzyme denaturation.
- Enzymes have an optimal temperature (usually around 37°C for human enzymes).
- pH:
- Optimal pH for amylase is around 6.7–7.0.
- At pH 3, activity drops significantly due to disruption of enzyme structure and active site.
- Enzyme Concentration:
- Increasing enzyme concentration increases reaction rate up to a point.
- Beyond that, the rate plateaus as substrate becomes limiting.
- b) Why Enzyme Activity Decreased at 60°C and pH 3:
- At 60°C:
The enzyme becomes denatured — its 3D structure breaks down, especially at the active site, reducing its ability to bind substrate. - At pH 3:
The acidic environment alters the ionic and hydrogen bonds that maintain enzyme shape, distorting the active site, leading to loss of activity.
Subsection: Coenzymes
Case Scenario 8:
Ramesh, a 22-year-old college student, visits the clinic with complaints of fatigue, glossitis (inflamed tongue), and numbness in hands and feet. His diet is mostly fast food and lacks fresh fruits and vegetables. Blood tests reveal megaloblastic anemia and low levels of Vitamin B12 and folic acid. The doctor explains that these vitamins are essential coenzymes involved in DNA synthesis and red blood cell formation.
- a) Define coenzymes and explain how they assist enzymes in biochemical reactions.
b) Mention the coenzymes derived from Vitamin B12 and folic acid, and state their main function.
c) Name any two other vitamins that act as coenzymes and mention one function of each.
Answer:
- a) Definition of Coenzymes and Their Role:
- Coenzymes are non-protein organic molecules that bind to enzymes and help in catalyzing biochemical reactions.
- They often act as carriers of electrons, atoms, or functional groups during the reaction.
- Coenzymes are usually derived from vitamins and are essential for enzyme activity.
- Without coenzymes, many enzymes would be inactive or inefficient.
- b) Coenzymes Derived from Vitamin B12 and Folic Acid:
| Vitamin | Coenzyme Form | Main Function |
| Vitamin B12 | Methylcobalamin | Transfers methyl groups; crucial for DNA synthesis and myelin formation. |
| Folic Acid | Tetrahydrofolate (THF) | Transfers one-carbon units in the synthesis of purines and thymidine, essential for DNA synthesis. |
- c) Two Other Vitamins Acting as Coenzymes:
| Vitamin | Coenzyme Form | Function |
| Vitamin B1 (Thiamine) | Thiamine pyrophosphate (TPP) | Coenzyme in carbohydrate metabolism (e.g., pyruvate dehydrogenase) |
| Vitamin B6 (Pyridoxine) | Pyridoxal phosphate (PLP) | Coenzyme in amino acid metabolism (e.g., transamination) |
Subsection: Enzyme Inhibition
Case Scenario 9:
John, a 45-year-old man, visited his doctor after getting his blood test results. He does not exercise much and eats a lot of fatty foods. His father had a heart attack at age 55. John’s blood tests showed high levels of total cholesterol and LDL cholesterol (“bad” cholesterol). His doctor diagnosed him with hypercholesterolemia (high cholesterol) and prescribed him a medicine called lovastatin.
- What is the mechanism of action of lovastatin?
- What is the difference between reversible and irreversible enzyme inhibition? Give suitable examples.
Answer:
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- a) Mechanism of Action of Lovastatin:
- Lovastatin is a competitive inhibitor of the enzyme HMG-CoA reductase.
- HMG-CoA reductase catalyzes the rate-limiting step in cholesterol biosynthesis in the liver.
- By inhibiting this enzyme, lovastatin:
- Reduces cholesterol synthesis.
- Increases LDL receptor expression on liver cells.
- Enhances uptake of LDL cholesterol from the blood, thereby lowering plasma cholesterol levels.
Summary: Lovastatin lowers cholesterol by inhibiting HMG-CoA reductase, a key enzyme in cholesterol production.
- b) Difference Between Reversible and Irreversible Enzyme Inhibition:
Feature Reversible Inhibition Irreversible Inhibition Binding Non-covalent (weak, reversible) Covalent (strong, permanent) Effect Temporary; enzyme can regain activity Permanent loss of enzyme activity Enzyme activity Can be restored after removal of inhibitor Cannot be restored; new enzyme must be synthesized Example Lovastatin (competitive reversible inhibitor of HMG-CoA reductase) Aspirin (irreversible inhibitor of COX enzyme)
Case Scenario 10:
Sandra, a 60-year-old woman, presents to her physician with complaints of recurrent blood clots in her legs, known as deep vein thrombosis (DVT). She has a history of atrial fibrillation and has previously experienced a stroke. Concerned about her risk of further thrombotic events, Sarah’s physician considers the use of anticoagulant therapy with dicumarol to prevent clot formation.
- Mention the mechanism of action of dicumarol in present
- Explain reversible and irreversible enzyme inhibition with suitable
Answer:
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- a) Mechanism of Action of Dicumarol:
Dicumarol is an oral anticoagulant that works by inhibiting the enzyme vitamin K epoxide reductase.
- This enzyme is essential for the regeneration of active vitamin K, which is needed for the γ-carboxylation of clotting factors II, VII, IX, and X.
- By inhibiting this enzyme, dicumarol reduces the synthesis of functional clotting factors, thus preventing blood clot formation.
- In Sandra’s case, it helps prevent recurrent deep vein thrombosis (DVT) and stroke by reducing the risk of abnormal clotting.
- b) Reversible and Irreversible Enzyme Inhibition:
Reversible Inhibition:
- Inhibitor binds to enzyme non-covalently.
- Effect can be reversed by removing the inhibitor.
- Example: Competitive inhibition – Sulfonamides inhibit dihydropteroate synthase by competing with PABA.
Irreversible Inhibition:
- Inhibitor binds to enzyme covalently, causing permanent inactivation.
- Enzyme activity cannot be restored unless new enzyme is synthesized.
- Example: Aspirin irreversibly inhibits cyclooxygenase (COX) enzyme in platelets, reducing thromboxane A₂ production.
Case Scenario 11:
Michael, a 10-year-old boy, is brought to the pediatric oncology clinic by his parents due to persistent fatigue, pallor, and easy bruising. His parents also notice swelling in his lymph nodes and abdomen. Michael’s medical history is unremarkable, and there is no family history of hematologic disorders. Initial blood tests reveal cytopenias and the presence of blast cells in his peripheral blood, raising suspicion for acute lymphoblastic leukemia (ALL). Physician advised methotrexate drug for the treatment.
- Mention the mechanism of action of methotrexate in above
- Explain reversible and irreversible enzyme inhibition with suitable
Answer:
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- a) Mechanism of Action of Methotrexate in ALL:
- Methotrexate is a structural analog of folic acid.
- It acts as a competitive inhibitor of the enzyme dihydrofolate reductase (DHFR).
- DHFR is required to convert dihydrofolate to tetrahydrofolate (THF), which is essential for purine and thymidylate synthesis, both of which are needed for DNA replication.
- By inhibiting DHFR, methotrexate:
- Blocks DNA synthesis in rapidly dividing cells (like leukemic blasts).
- Leads to cell cycle arrest and cell death (especially in S-phase cells).
Summary: Methotrexate inhibits DHFR, blocking DNA synthesis and targeting rapidly dividing cancer cells in ALL.
- b) Reversible vs. Irreversible Enzyme Inhibition (with Examples):
Feature Reversible Inhibition Irreversible Inhibition Binding Weak, non-covalent binding to the enzyme Strong, covalent binding to the enzyme Effect Temporary; enzyme regains function when inhibitor is removed Permanent; enzyme function is lost Enzyme Activity Can be restored Cannot be restored; new enzyme must be synthesized Example Methotrexate – reversible inhibitor of DHFR Aspirin – irreversible inhibitor of COX enzyme
Case Scenario 12:
David, a 55-year-old man, presents to his primary care physician with complaints of severe pain, swelling, and redness in his right big toe. He recalls similar episodes in the past, typically triggered by dietary indiscretion or alcohol consumption. David works as a chef and enjoys cooking rich, high-purine foods. His family history is significant for gout, with his father and older brother experiencing similar symptoms. Physician advised Allopurinol drug for the treatment.
- Mention the mechanism of action of allopurinol in above
- Explain competitive and non-competitive enzyme inhibition with suitable
Answer:
- Mechanism of Action of Allopurinol:
- Allopurinol is a structural analog of hypoxanthine.
- It acts as a competitive inhibitor of the enzyme xanthine oxidase.
- Xanthine oxidase catalyzes the conversion of:
- Hypoxanthine → Xanthine
- Xanthine → Uric acid
- By inhibiting this enzyme, allopurinol:
- Reduces uric acid production
- Prevents uric acid crystal formation in joints
- Helps in long-term management of chronic gout
Summary: Allopurinol competitively inhibits xanthine oxidase, reducing uric acid synthesis and preventing gout attacks.
- b) Competitive vs. Non-Competitive Enzyme Inhibition (with Examples):
Feature Competitive Inhibition Non-Competitive Inhibition Binding site Inhibitor binds to active site of the enzyme Inhibitor binds to allosteric site (not the active site) Effect on substrate binding Blocks substrate from binding Substrate can still bind, but enzyme activity is reduced Reversibility Usually reversible by increasing substrate concentration Not reversed by adding more substrate Example Allopurinol – competitive inhibitor of xanthine oxidase Lead – non-competitive inhibitor of ferrochelatase
Subsection: Enzyme Specificity
Case Scenario 13:
A 19-year-old MBBS student is performing an experiment in the biochemistry lab to study enzyme action. She is provided with three test tubes containing urea, ammonium chloride, and guanidine. She adds the enzyme urease to each of the test tubes and observes that only the test tube with urea shows the release of ammonia and a change in pH. The other test tubes show no reaction.
- What does this experiment indicate about the enzyme urease? Explain the concept of absolute enzyme specificity using this case.
- Differentiate between absolute specificity and other types of enzyme specificity with suitable examples.
Answer:
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- What Does This Experiment Indicate About Urease?
The experiment shows that urease acts only on urea and not on similar molecules like ammonium chloride or guanidine.
This demonstrates the enzyme’s absolute specificity, meaning it catalyzes the reaction only for one specific substrate — urea in this case.
Urease catalyzes:
Urea → Ammonia + Carbon dioxideAbsolute Enzyme Specificity (with this case):
Definition: When an enzyme acts only on one specific substrate, it exhibits absolute specificity.
In this case:
Urease acts only on urea → shows absolute specificity.
No reaction occurs with structurally similar compounds → confirms specificity.
- Types of Enzyme Specificity (with Examples):
Type of Specificity Definition Example Absolute Specificity Enzyme acts only on one substrate Urease acts only on urea Group Specificity Acts on molecules with a specific functional group Alcohol dehydrogenase acts on alcohols Linkage Specificity Acts on a particular type of bond, regardless of rest of molecule Proteases cleave peptide bonds Stereochemical Specificity Acts only on a particular stereoisomer Lactate dehydrogenase acts on L-lactate