Holoenzyme = Apoenzyme (protein) + Cofactor (non-protein)
A: Coenzyme = organic cofactor (loosely bound, e.g., NAD+)
B: Cofactor = general term for non-protein component
C: Apoenzyme = protein component of holoenzyme ✓
D: Prosthetic group = tightly bound organic cofactor
Answer: Apoenzyme
Enzymes named based on: substrate + -ase suffix (amylase, protease, lipase), reaction type (oxidoreductase, transferase), or trivial names (pepsin, trypsin). IUB (International Union of Biochemistry) classification: 6 major classes. EC1 Oxidoreductases: oxidation-reduction (dehydrogenases, oxidases, reductases). EC2 Transferases: transfer functional groups (kinases, transaminases). EC3 Hydrolases: hydrolysis (proteases, lipases, amylases, nucleases). EC4 Lyases: addition/removal of groups to/from double bonds (decarboxylases, dehydratases). EC5 Isomerases: isomerisation (isomerases, mutases, epimerases). EC6 Ligases: form bonds using ATP (synthetases, carboxylases). EC number: 4 digits (e.g., hexokinase = EC 2.7.1.1).
Active site: region of enzyme where substrate binds and reaction occurs. Specific 3D shape (complementary to substrate). Typically 3-12 amino acids out of hundreds. Lock and key model (Fischer 1894): rigid complementarity. Active site shape exactly fits substrate. Explains specificity but not flexibility. Induced fit model (Koshland 1958): enzyme undergoes conformational change when substrate binds. Active site moulds around substrate. Better explains: broad substrate specificity of some enzymes, role of cofactors, allosteric regulation. Transition state theory: enzyme binds transition state better than substrate or product. Stabilises transition state → lowers activation energy. Binding energy: multiple weak interactions (H-bonds, van der Waals, ionic, hydrophobic) between enzyme and substrate provide binding energy for catalysis.
Michaelis-Menten equation: v = Vmax[S]/(Km + [S]). Km (Michaelis constant): [S] at half-Vmax. Measure of enzyme-substrate affinity. Low Km = high affinity. Vmax = maximum velocity (all enzyme saturated). kcat = turnover number = reactions per enzyme per second = Vmax/[E]total. kcat/Km = catalytic efficiency. Lineweaver-Burk (double reciprocal plot): 1/v vs 1/[S]. Linear. x-intercept = -1/Km. y-intercept = 1/Vmax. Inhibition types: Competitive: inhibitor resembles substrate, binds active site. Increases apparent Km (more substrate needed to outcompete inhibitor). Vmax unchanged. Non-competitive: inhibitor binds allosteric site (not active site). Reduces Vmax. Km unchanged. Uncompetitive: inhibitor binds enzyme-substrate complex only. Decreases both Km and Vmax. Mixed inhibition: changes both Km and Vmax.
Allosteric enzymes: have multiple binding sites. Regulatory site (allosteric site) separate from active site. Conformational change upon allosteric binding affects active site activity. Positive allosteric effector: activates enzyme (increases affinity or kcat). Negative allosteric effector: inhibits enzyme. Sigmoid (S-shaped) velocity curve (cooperativity). Hill equation: v = Vmax[S]^n/(K0.5^n + [S]^n). n = Hill coefficient. n > 1: positive cooperativity. n = 1: no cooperativity (Michaelis-Menten). n < 1: negative cooperativity. Examples: haemoglobin (O2 binding cooperative), phosphofructokinase-1 (activated by AMP/ADP, inhibited by ATP, citrate), aspartate transcarbamoylase (ATCase, inhibited by CTP = feedback inhibition). Feedback inhibition: end product of metabolic pathway inhibits enzyme early in pathway. Self-regulation. Conserves energy and resources.
B vitamins: essential coenzymes. B1 (Thiamine): TPP (thiamine pyrophosphate). Pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, transketolase. Deficiency: beriberi (peripheral neuropathy, heart failure), Wernicke encephalopathy. B2 (Riboflavin): FAD, FMN. Oxidoreductases. Deficiency: ariboflavinosis (glossitis, cheilosis). B3 (Niacin): NAD+, NADP+. >400 enzyme reactions (mainly redox). Deficiency: pellagra (dermatitis, diarrhoea, dementia, death = 4 Ds). B5 (Pantothenic acid): CoA (coenzyme A). Central metabolism (pyruvate entry into Krebs, fatty acid synthesis). B6 (Pyridoxine): PLP (pyridoxal phosphate). Transaminations (amino acid metabolism). Deficiency: seizures in neonates. B7 (Biotin): covalently bound to carboxylases. CO2 carrier. Deficiency: dermatitis (raw egg white contains avidin - binds biotin). B9 (Folate): THF (tetrahydrofolate). One-carbon transfer reactions. Nucleotide synthesis. Deficiency: megaloblastic anaemia, neural tube defects. B12 (Cobalamin): methylcobalamin, adenosylcobalamin. Methionine synthesis, odd-chain fatty acid metabolism. Deficiency: pernicious anaemia, subacute combined degeneration of cord.
Competitive inhibitors as drugs: Statins (HMG-CoA reductase inhibitors, cholesterol synthesis): atorvastatin, rosuvastatin. Competitive with HMG-CoA. ACE inhibitors (angiotensin-converting enzyme, blood pressure): lisinopril, enalapril. Competitive. Methotrexate (dihydrofolate reductase inhibitor, cancer/autoimmune): anti-folate, competes with dihydrofolate. Sulphonamide antibiotics (PABA analogue, competitive inhibitor of dihydropteroate synthase in bacterial folate synthesis). Non-competitive inhibitors: Heavy metals (Pb2+, Hg2+, As3+): bind -SH groups. Cyanide: binds Fe3+ in cytochrome c oxidase (Complex IV) = irreversible non-competitive inhibition. Irreversible inhibitors: organophosphates (nerve agents, insecticides): covalently modify serine at active site of acetylcholinesterase. Aspirin: acetylates serine in cyclooxygenase (COX-1, COX-2) active site. Irreversible inhibition of prostaglandin synthesis. Suicide inhibitors (mechanism-based): modified substrate that reacts with enzyme active site permanently.
Isoenzymes (isozymes): different molecular forms of the same enzyme, catalyse same reaction, differ in structure and properties. LDH (lactate dehydrogenase): 5 isoenzymes (LDH1-5). LDH1 (H4): heart. LDH5 (M4): liver and skeletal muscle. LDH2 (H3M1), LDH3 (H2M2), LDH4 (HM3). H = heart subunit (high affinity for lactate). M = muscle subunit (high affinity for pyruvate). After myocardial infarction: LDH1 rises. Serum protein electrophoresis detects. Now largely replaced by troponin. Creatine kinase (CK): CK-MB (heart specific, rises in MI). CK-BB (brain). CK-MM (skeletal muscle). Aldolase: A form (muscle), B form (liver), C form (brain). Clinical significance: isoenzyme patterns used for diagnosis (organ-specific damage releases tissue-specific isoenzymes into blood). ALT, AST: liver damage markers. Alkaline phosphatase: bone and liver isoforms.
Industrial enzymes: amylase (starch hydrolysis for glucose/fructose production, brewing), protease (detergents for stain removal, cheese making), lipase (biofuels, food processing), cellulase (biofuels, textile processing), glucose isomerase (HFCS production from glucose), rennin/chymosin (cheese making). Medical enzymes: streptokinase, tissue plasminogen activator (tPA): dissolve blood clots (thrombolysis in MI, stroke). Lysozyme: antibacterial, in wound care. Restriction enzymes: DNA cutting tools for molecular biology. DNA polymerase (Taq, Pfu): PCR. Reverse transcriptase: cDNA synthesis. DNA ligase: joining DNA fragments. Enzyme-linked immunosorbent assay (ELISA): enzyme-linked antibodies detect antigens quantitatively. Biosensors: immobilised enzymes detect analytes (glucose biosensor: glucose oxidase + electrode measures H2O2 produced).