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Topics: Classification, Co-enzymes
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Enzymes are classified by the International Union of Biochemistry (IUB) into six major classes based on the reactions they catalyze.
• Catalyze oxidation–reduction reactions.
• Transfer of electrons or hydrogen atoms.
• Examples: Dehydrogenases, oxidases, reductases.
• Transfer functional groups (methyl, amino, phosphate).
• Examples: Transaminases, kinases, methyltransferases.
• Catalyze hydrolysis of bonds (using water).
• Examples: Proteases, lipases, amylases, phosphatases.
• Break bonds without hydrolysis or oxidation, forming double bonds.
• Examples: Decarboxylases, aldolases.
• Catalyze intramolecular rearrangements.
• Examples: Racemases, epimerases, mutases.
• Join two molecules together using ATP.
• Examples: Carboxylase, DNA ligase.
O-T-H-L-I-L → “Only Tigers Hunt Lions In Laos”
• Simple enzymes → made of protein only.
• Conjugated enzymes → protein (apoenzyme) + non-protein part (cofactor).
– Apoenzyme + cofactor → holoenzyme.
• Intracellular enzymes → metabolic enzymes.
• Extracellular enzymes → digestive enzymes (amylase, lipase).
• Constitutive enzymes → always present.
• Inducible enzymes → upregulated when substrate appears (e.g., β-galactosidase).
Co-enzymes are organic, non-protein molecules required by some enzymes for catalytic activity.
Most are derived from vitamins.
• Participate in oxidation–reduction reactions.
• Accept hydride ions (H⁻).
• Used by dehydrogenases (e.g., lactate dehydrogenase).
• Accept two hydrogens in redox reactions.
• Cofactor for succinate dehydrogenase.
• Carries acyl groups.
• Essential for fatty acid oxidation, Krebs cycle, cholesterol synthesis.
• Coenzyme for transamination, decarboxylation, deamination.
• Used by aminotransferases (ALT, AST).
• Cofactor for carboxylation reactions.
• Enzymes: pyruvate carboxylase, acetyl-CoA carboxylase.
• Transfers one-carbon units (methyl, formyl).
• Essential for nucleotide synthesis.
• Required for methionine synthase and methylmalonyl-CoA mutase.
• Coenzyme in oxidative decarboxylation (PDH, α-ketoglutarate dehydrogenase).
• Also for transketolase in HMP shunt.
• Electron carrier in the electron transport chain.
• Cofactor in cytochromes & peroxidases.
• Coenzyme for pyruvate dehydrogenase and α-ketoglutarate dehydrogenase.
• Coenzymes → loosely bound, dissociable.
• Prosthetic groups → tightly or covalently attached.
• B-complex vitamin deficiency → enzyme dysfunction → metabolic disorders.
• ALT/AST require PLP; deficiency → defective amino-acid metabolism.
• B₁ deficiency → PDH dysfunction → lactic acidosis.
• Enzymes speed up reactions by lowering activation energy (Ea).
• They do NOT change ΔG (free energy) or equilibrium constant.
• Substrate binds to the enzyme’s active site → forms ES complex.
• ES complex stabilizes the transition state → faster product formation.
• Lock and Key Model
– Active site is rigid and fits substrate exactly.
• Induced Fit Model
– Active site is flexible; binding induces conformational change.
– More accurate for most enzymes.
E + S → ES → EP → E + P
• Enzyme remains unchanged after reaction.
• The region of the enzyme where substrate binding and catalysis occur.
• Occupies only a small portion of the enzyme.
• Formed by specific amino acids (Ser, His, Asp, Cys, Lys, Glu).
• 3D orientation determines specificity.
• Binding residues → hold the substrate.
• Catalytic residues → perform bond-breaking/bond-making.
• Hydrophobic pocket
• Correct orientation for catalysis
• Stabilizes transition state
• Absolute (urease acts only on urea)
• Group-specific (hexokinase phosphorylates many hexoses)
• Stereo-specific (L-amino acid oxidase)
v = (Vmax × [S]) / (Km + [S])
• v = reaction velocity
• Vmax = maximum velocity when enzyme is saturated
• Km = substrate concentration at ½ Vmax
• ES complex formation is reversible.
• Steady-state concentration of ES.
• Substrate >> enzyme concentration.
• At low [S] → reaction first-order (rate ∝ [S]).
• At high [S] → reaction zero-order (rate independent of [S]).
• Vmax depends on enzyme concentration.
• Km is independent of enzyme concentration.
• Km is the substrate concentration at which the reaction velocity is half of Vmax.
• Measures enzyme affinity for substrate.
– Low Km → high affinity → enzyme saturates quickly
– High Km → low affinity
• GIVES a quantitative measure of how strongly an enzyme binds its substrate.
• Hexokinase has low Km → high affinity → active even at low glucose.
• Glucokinase has higher Km → active only after meals → prevents hypoglycemia.
• Useful in diagnosing genetic enzyme defects.
• Enzymes synthesized in inactive precursor forms (zymogens).
• Activated by proteolytic cleavage.
Examples:
• Pepsinogen → pepsin
• Trypsinogen → trypsin
• Activator binds to allosteric site → increases enzyme activity.
Example:
• ATP activates phosphofructokinase-1 (PFK-1) in glycolysis (at high energy states).
• Phosphorylation/dephosphorylation alters enzyme activity.
Examples:
• Glycogen phosphorylase active when phosphorylated.
• Acetyl-CoA carboxylase active when dephosphorylated.
• Some enzymes require metal ions as activators.
Examples:
• Mg²⁺ → kinases
• Zn²⁺ → carbonic anhydrase
• Ca²⁺ → clotting enzymes
• Each enzyme has optimum pH & temperature.
• Small changes can enhance activity until denaturation occurs.
• In competitive inhibition, the inhibitor resembles the substrate and competes for the active site of the enzyme.
• Inhibitor binds only to the active site of free enzyme (E).
• Prevents ES complex formation.
• Reversible by increasing substrate concentration.
• Vmax → unchanged
• Km → increased (lower affinity, more substrate needed)
• Lineweaver–Burk Plot:
– Lines intersect on the y-axis (same Vmax).
– Slope increases.
• Malonate inhibits succinate dehydrogenase.
• Statins competitively inhibit HMG-CoA reductase.
• Methotrexate inhibits dihydrofolate reductase.
• Increasing substrate (e.g., high-dose folate) can overcome methotrexate toxicity.
• Inhibitor binds to a site other than the active site (allosteric site).
• Binding distorts enzyme conformation → reduces activity.
• Inhibitor can bind to E or ES complex.
• Does not compete with substrate.
• Cannot be reversed by increasing substrate concentration.
• Vmax → decreased
• Km → unchanged (affinity same, but active enzyme molecules fewer)
• Lineweaver–Burk Plot:
– Lines intersect on the x-axis (same Km).
– Slope increases, y-intercept increases.
• Cyanide inhibits cytochrome oxidase.
• Heavy metals (Hg²⁺, Ag⁺) inhibit SH-containing enzymes.
• Alanine noncompetitively inhibits pyruvate kinase.
• Removal of inhibitor or chelation of metal ions can restore activity (e.g., BAL for arsenic poisoning).
• Allosteric inhibition occurs when an inhibitor binds to an allosteric (regulatory) site, not the active site.
• Binding causes a conformational change → decreased enzyme activity.
• Does not resemble the substrate.
• Can act rapidly and reversibly.
• Often occurs in regulatory enzymes of metabolic pathways.
• Shows sigmoidal (S-shaped) kinetics, not Michaelis–Menten.
• ATP inhibits phosphofructokinase-1 (PFK-1) in glycolysis.
• CTP inhibits aspartate transcarbamoylase.
• Enzymes that catalyze rate-limiting steps of metabolic pathways.
• Usually allosteric enzymes.
• Irreversible, early in the pathway.
• Highly regulated by activators/inhibitors.
• PFK-1 – rate-limiting enzyme of glycolysis
• Glutamate dehydrogenase – amino-acid metabolism
• HMG-CoA reductase – cholesterol synthesis
• Glycogen phosphorylase – glycogen breakdown
• Carbamoyl phosphate synthetase I – urea cycle
• Acetyl-CoA carboxylase – fatty acid synthesis
• End-product of a metabolic pathway inhibits the first committed step → prevents overproduction.
• End-product binds to an allosteric site of the initial enzyme.
• Reduces enzyme activity by conformational change.
• Maintains metabolic balance.
• Prevents waste of energy and substrates.
• Quick and reversible control mechanism.
• Isoleucine inhibits threonine dehydratase.
• Cholesterol inhibits HMG-CoA reductase.
• ATP inhibits PFK-1.
• Inhibitor binds only to the ES complex, not to free enzyme.
• Prevents formation of product → ES becomes ESI (inactive).
• Vmax → decreased
• Km → decreased
(Because inhibitor locks ES complex, making enzyme appear to have higher affinity)
• Cannot be reversed by increasing substrate concentration.
• Lines are parallel (same slope).
• Y-intercept increases; x-intercept shifts.
• Lithium inhibits inositol monophosphatase uncompetitively.
• Double reciprocal plot used to determine Km and Vmax and to differentiate types of inhibition.
1/v = (Km/Vmax) × (1/[S]) + 1/Vmax
• Straight line where:
– Y-intercept = 1/Vmax
– X-intercept = –1/Km
– Slope = Km/Vmax
1. Competitive Inhibition
• Vmax same
• Km increases
• Lines intersect at y-axis
2. Noncompetitive Inhibition
• Vmax decreases
• Km unchanged
• Lines intersect at x-axis
3. Uncompetitive Inhibition
• Vmax decreases
• Km decreases
• Lines are parallel
• Easy comparison of inhibition patterns.
• Distorts error at low substrate concentrations; Eadie–Hofstee plot is more accurate.
• Regulation of enzyme activity through reversible covalent addition or removal of a chemical group.
• Kinases add phosphate (ATP → ADP).
• Phosphatases remove phosphate.
• Can activate or inhibit depending on the enzyme.
• Glycogen phosphorylase → active when phosphorylated.
• Glycogen synthase → inactive when phosphorylated.
• Acetyl-CoA carboxylase → active when dephosphorylated.
• Adenylation
• Methylation
• ADP-ribosylation
• Ubiquitination (→ marks proteins for degradation)
• Long-term regulation where synthesis of an enzyme is suppressed at the gene level when its product is abundant.
• Slower, affects amount of enzyme, not immediate activity.
• Seen in bacteria and human metabolic pathways.
• High cholesterol represses HMG-CoA reductase gene expression.
• Increased gene expression → increased enzyme synthesis in response to a metabolite or drug.
• High carbohydrate diet induces glucokinase.
• Barbiturates induce cytochrome P450 enzymes.
• Lactose induces β-galactosidase in bacteria.
• Allows metabolic adaptation to environmental or dietary conditions.
• Activity increases with temperature up to optimum (~37°C).
• High temperature → denaturation.
• Each enzyme has an optimum pH.
• Extreme pH → denatures enzyme.
• Activity increases until Vmax is reached (enzyme saturation).
• Follows the Michaelis–Menten curve.
• Rate ∝ enzyme concentration (when substrate is in excess).
• Accumulation of product slows reaction (product inhibition).
• Metal ions (Mg²⁺, Zn²⁺, Ca²⁺) often essential.
• Example: kinases need Mg²⁺.
• Competitive, noncompetitive, uncompetitive, allosteric inhibitors decrease activity.
• Different molecular forms of the same enzyme that catalyze the same reaction but differ in structure, kinetics, and tissue distribution.
• Useful in diagnosing tissue damage because each isoenzyme is tissue-specific.
LDH has five isoenzymes (tetramers of H and M subunits):
LDH-1 (H4) – Heart, RBC
LDH-2 (H3M1) – Reticuloendothelial system
LDH-3 (H2M2) – Lungs
LDH-4 (H1M3) – Kidneys, pancreas
LDH-5 (M4) – Liver, skeletal muscle
• MI (heart attack) → LDH-1 ↑ above LDH-2 (flipped pattern).
• Liver disease / muscle injury → LDH-5 ↑.
• Hemolysis → LDH-1 ↑ (released from RBCs).
CK exists in three isoforms:
CK-BB (CK-1)
• Brain, smooth muscle
• Increased in CNS injury
CK-MB (CK-2)
• Heart muscle
• Most specific marker for myocardial infarction
• Rises 4–6 hours after MI, peaks at 24 hours, normal in 48 hours
CK-MM (CK-3)
• Skeletal muscle
• Increased in muscular dystrophy, rhabdomyolysis, trauma
• LDH isoenzymes → differentiate liver, heart, lung, muscle diseases.
• CK-MB → early diagnosis of acute myocardial infarction.
• CK-BB → stroke, CNS tumors.
• CK-MM → muscle injury.
Enzymes show high specificity toward substrates and reactions.
• Enzyme acts only on one substrate.
• Example: Urease → only urea.
• Acts on substrates with similar functional groups.
• Example: Hexokinase → phosphorylates many hexoses.
• Acts only on a particular type of bond.
• Example: Esterases → hydrolyze ester bonds.
• Distinguish between D- and L-forms.
• Example: L-amino acid oxidase, D-lactate dehydrogenase.
• One type of chemical transformation only.
• Example: Oxidoreductases → redox reactions only.
Modification of enzymes through biochemical, genetic, or structural changes to improve function.
• Site-directed mutagenesis → change specific amino acids.
• Directed evolution → repeated mutation + selection.
• Fusion proteins → catalytic domain + tag (His-tag).
• Improved stability (heat-stable enzymes).
• Reduced inhibition.
• Faster industrial biocatalysis (detergent enzymes).
• Design of insulin analogs, engineered proteases, and enzyme replacement therapies.
• Amount of enzyme that converts 1 micromole of substrate per minute under defined conditions.
• SI unit.
• Amount converting 1 mole of substrate per second.
• (1 katal = 60,000 IU)
• Units of enzyme per mg of protein.
• Indicates enzyme purity.
• Number of substrate molecules converted to product per enzyme molecule per second.
(Already partly covered earlier, expanded here)
Different molecular forms of the same enzyme with:
• same catalytic action,
• different amino acid sequences,
• different tissue distribution.
• LDH (LDH-1 → LDH-5)
• Creatine Kinase (CK-BB, CK-MB, CK-MM)
• Alkaline phosphatase (ALP) isoenzymes – liver, bone, placenta
• Amylase – pancreatic vs salivary
Enzymes used as biomarkers for tissue injury.
• CK-MB → MI (rises 4–6 h, normal in 48 h)
• LDH-1 → MI (LDH1 > LDH2 = flipped pattern)
• Troponin (not an enzyme but key marker)
• ALT (SGPT) → hepatocellular damage
• AST (SGOT) → liver & muscle
• ALP → cholestasis, bone disease
• GGT → alcoholism, biliary obstruction
• Amylase
• Lipase → more specific for acute pancreatitis
• CK-MM → muscle injury, rhabdomyolysis
• Aldolase → muscle diseases
• Bone ALP → rickets, Paget disease
• Placental ALP → pregnancy, germ cell tumors
Separation of isoenzymes based on differences in charge, mobility, and size.
• Agarose gel electrophoresis
• Cellulose acetate electrophoresis
• Isoelectric focusing
LDH isoforms migrate differently:
• LDH-1 (H4) → fastest, most negative, moves furthest
• LDH-5 (M4) → slowest, least negative
• Diagnosing myocardial infarction (LDH1 > LDH2).
• Differentiating liver vs bone ALP.
• Identifying cancer-related isoenzyme patterns.
• Confirming salivary vs pancreatic amylase.
• Enzymes show absolute, group, stereo, bond specificity.
• Enzyme engineering modifies catalytic efficiency and stability.
• IU = amount of enzyme converting 1 μmol/min.
• Isoenzymes differ in structure but catalyze same reaction.
• LDH-1 elevation → myocardial infarction.
• CK-MB → most specific enzymatic marker for MI.
• ALP high with GGT normal → bone disease.
• Electrophoresis separates isoenzymes based on charge differences.
The 3D structure of the active site, which recognizes the substrate based on shape, charge, and stereochemistry.
Enzyme acts on only one substrate. Example: Urease → Urea.
Enzyme acts on substrates with similar functional groups (e.g., hexokinase).
To improve enzyme stability, activity, or specificity using genetic or chemical modifications.
A technique to modify specific amino acids in an enzyme to alter function.
Amount of enzyme that converts 1 micromole of substrate per minute.
SI unit of enzyme activity = 1 mole of product per second.
Units of enzyme per mg of protein—an indicator of enzyme purity.
Different molecular forms of an enzyme, with same function but different structure and tissue distribution.
They help identify which tissue is damaged during disease.
LDH-1 > LDH-2 (“flipped pattern”).
CK-MB.
Rises at 4–6 hours, peaks at 24 hours, normal in 48 hours.
Amylase and lipase, with lipase being more specific.
↑ ALT, ↑ AST (AST may rise higher in alcohol-related damage).
↑ ALP and ↑ GGT.
Rickets, osteomalacia, Paget disease.
CK-MM.
By electrophoresis (agarose gel, cellulose acetate) or isoelectric focusing.
Pancreatic amylase.
Reduction in enzyme synthesis at gene level when product is abundant.
Increased enzyme synthesis in response to a substrate, hormone, or drug.
Cytochrome P450 enzymes induced by barbiturates.
Mg²⁺.
It distinguishes tissue-specific enzyme forms, aiding diagnosis (heart vs liver vs bone pathology).
A. Hexokinase
B. Trypsin
C. Urease
D. Lipase
Answer: C
A. Absolute
B. Group
C. Bond
D. Reaction
Answer: B
A. Pepsin
B. D-amino acid oxidase
C. Amylase
D. Catalase
Answer: B
A. Enzyme repression
B. Enzyme engineering
C. Feedback inhibition
D. Zymogen activation
Answer: B
A. 1 mmol/min
B. 1 μmol/min
C. 1 mol/sec
D. 1 μmol/sec
Answer: B
A. Purity of enzyme
B. pH of enzyme
C. Amount of substrate
D. Temperature stability
Answer: A
A. LDH-5
B. LDH-3
C. LDH-1
D. LDH-4
Answer: C
A. Forward pattern
B. Flipped pattern
C. Reverse pattern
D. Saturation pattern
Answer: B
A. Liver failure
B. Acute pancreatitis
C. Skeletal muscle injury
D. Myocardial infarction
Answer: D
A. LDH
B. CK-MB
C. Troponin I
D. AST
Answer: B
A. ALT
B. AST
C. ALP
D. LDH
Answer: C
A. Acute bone disease
B. Alcoholic liver disease
C. Muscle injury
D. Rickets
Answer: B
A. Amylase
B. Trypsin
C. Lipase
D. Elastase
Answer: C
A. Cirrhosis
B. Paget disease
C. Myocardial infarction
D. Cushing syndrome
Answer: B
A. Heart
B. Skeletal muscle
C. Brain
D. Liver
Answer: C
A. Function
B. Activation energy
C. Amino acid sequence
D. Reaction catalyzed
Answer: C
(Reaction catalyzed is same.)
A. Simple centrifugation
B. Electrophoresis
C. Precipitation
D. Dialysis
Answer: B
A. Km increases
B. Km decreases
C. Vmax decreases
D. Vmax increases
Answer: C
A. A simple enzyme
B. An allosteric enzyme
C. A hydrolase
D. A zymogen
Answer: B
A. The last enzyme of the pathway
B. Any random enzyme
C. The rate-limiting enzyme
D. The fastest enzyme
Answer: C
A. Competitive
B. Non-competitive
C. Uncompetitive
D. Allosteric
Answer: C
A. Pepsin
B. Kinases
C. Urease
D. Lipase
Answer: B
A. Increase in substrate concentration
B. Increase in enzyme activity
C. Increase in enzyme synthesis
D. Decrease in enzyme affinity
Answer: C
A. Vitamin C
B. Barbiturates
C. Insulin
D. Iron deficiency
Answer: B
A. Substrate is in excess
B. Product accumulates
C. Temperature increases
D. pH increases
Answer: B
It is the ability of an enzyme to choose a single substrate or group of substrates based on its active-site structure.
The enzyme acts on only one specific substrate.
Example: Urease → urea.
The enzyme catalyzes reactions of substrates with similar functional groups.
Example: Hexokinase.
The enzyme distinguishes between D- and L-forms of molecules.
The enzyme catalyzes only one type of chemical reaction, regardless of substrate variety.
Modification of enzyme structure by genetic or chemical methods to improve activity, stability, or specificity.
Site-directed mutagenesis to create heat-stable enzymes.
Technique to change a specific amino acid in a protein to alter its function.
Amount of enzyme that converts 1 μmol of substrate per minute.
SI unit of enzyme activity (1 mole of substrate converted per second).
Enzyme units per mg of protein—indicator of enzyme purity.
The number of substrate molecules converted per enzyme molecule per second.
Different molecular forms of the same enzyme with different structures but identical catalytic function.
They help identify which tissue is damaged, since each isoenzyme is tissue-specific.
LDH-1, showing the flipped pattern (LDH-1 > LDH-2).
CK-MB.
Rises in 4–6 hours, peaks at 24 hours, normal after 48 hours.
Lipase.
ALP, along with GGT.
Bone ALP.
Skeletal muscle injury or rhabdomyolysis.
A technique to separate isoenzymes based on charge and mobility.
Isoelectric focusing, based on pI differences.
The end-product inhibits the rate-limiting enzyme of its own pathway.
Decreased enzyme synthesis at the gene level due to excess product.
Increased enzyme synthesis in response to a metabolite, hormone, or drug.
Sigmoidal kinetics and rapid, reversible control.
ATP.
Mg²⁺.
Obstructive or alcoholic liver disease.
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