Lecture 4 Enzymes PDF

4.1 Structure and Components of Enzymes Measure of enzyme activity - Expressed as international unit (IU): catalyses conversion of 1 µmol of substrate to product per minute - specific activity of enzyme = IU/mg protein Classification of enzymes (= 7 types of enzymes) 1. Oxidored...

4.1 Structure and Components of Enzymes Measure of enzyme activity - Expressed as international unit (IU): catalyses conversion of 1 µmol of substrate to product per minute - specific activity of enzyme = IU/mg protein Classification of enzymes (= 7 types of enzymes) 1. Oxidoreductases Ared+ Box→Aox+ Bred - Transfer of e- (H atoms) 2. Transferases A–B + C → A + B–C - Group transfer reactions (except H atoms) 3. Hydrolases A–B + H2O → A–H + B–OH - Catalyse hydrolysis reaction with addition of H2O 4. Lyases/synthases X-A—B-Y→ A=B + X-Y - Cleavage of bonds by elimination → leaving double bonds or ring structures - Addition of groups to double bonds 5. Isomerases A ⇌ isoA - Transfer to groups within molecules to yield isomeric forms 6. Ligases A + B + ATP → A–-B + ADP + Pi - Formation of bonds by condensation reactions coupled to cleavage of ATP 7. Translocases - Assists in moving another molecule, usually across a cell membrane Reaction and substrate specificity - Mostly highly specific for type of reaction catalysed and nature of substrate ○ eg catalase for H2O2 , urease for urea - Others have broader substrate specificity ○ eg trypsin for catalysing digestion of peptide bonds Active site (= cleft for catalysis) - Contains residues that directly participate in making/breaking of bonds - Binds with substrate (may require cofactor/coenzyme) 4.2 Roles of coenzymes in enzymatic reactions Cofactors and coenzymes (= helper molecules for enzymes) - Cofactors = inorganic elements (eg Ca2+, Fe, Cu) ○ Minerals as cofactors for enzymes - Coenzyme = organic molecules (eg CoA) ○ Usually modified during enzymatic reaction and recycled by another enzyme ○ May assist in transport of intermediates during sequence of reactions → act as transient carriers for functional groups ○ Most vitamins are precursors of coenzymes - Can be loosely and transiently bound to enzymes - Can also be tightly and permanently bound to enzymes as prosthetic groups (eg heme group) EXAMPLE: vitamin B6 and coenzyme derivatives - Pyridoxal phosphate (form of vitamin B6): required in AA synthesis, catabolism and interconversion ○ Acts as transient carrier of amino group during AA metabolism - B6 requirement increases with protein intake ○ Present as supplements in formula milk → promotes effective protein synthesis ○ B6 deficiency: neurologic symptoms and anaemia 4.3 Mechanisms regulating enzymatic reactions Working mechanism of enzyme - Active site of enzyme binds with substrate → catalysis of reaction - Enzymes lower activation energy for transition state formation ○ ΔGB (binding energy) = free energy that is released by formation of weak interactions between ES complex ○ Enzymes increase reaction rate, but not the eqm of reaction Enzyme kinetics 1. Michaelis-Menten kinetics [𝑆] 𝑉0 = 𝑉𝑚𝑎𝑥 𝐾𝑚+[𝑆] - When [S] tends to ∞ ; V0 approaches Vmax - Vmax occurs when enzyme is saturated, where [ES] = [Etotal] When Km = [S], then 1 𝑉0 = 2 𝑉𝑚𝑎𝑥 - Km = substrate conc where the initial reaction rate is half of that its maximal rate ○ dissociation constant for ES → E+S = binding affinity of enzyme for its substrates ○ Km has the same unit as [S] - ↓ Km of enzyme = ↑ affinity of enzyme for substrate (vice versa) 1 ○ Requires less [S] to reach 2 𝑉𝑚𝑎𝑥 = ↑ affinity to substrate  2. Lineweaver-Burk double reciprocal plot By taking reciprocal on both sides, we have: 1 𝐾𝑚 1 1 𝑉0 = 𝑉𝑚𝑎𝑥 · [𝑆] + 𝑉𝑚𝑎𝑥 - Concept of linear equation (y = mx + c) Given that k2 = rate constant of ES → E + P, 𝑉𝑚𝑎𝑥 𝑘2 = [𝐸𝑡𝑜𝑡𝑎𝑙] - If ES → E + P is a rate-limiting reaction, then k2 = Kcat (turnover number) ○ Kcat = number of substrate molecules bring converted to product per unit time when enzyme is saturated with substrate ○ Unit = s-1 𝐾𝑐𝑎𝑡 - To measure catalytic efficiency, we have 𝐾 : 𝑚 ○ Km = determines first step (E+S → ES) ○ Kcat = determines second step (ES → E+P) 4.4 Nature and inhibition of enzymes Competitive inhibition (reversible) - Competes with substrate for active site → effect can be “diluted” by adding more substrate - Does not inactivate enzyme - Structurally similar to substrates - Vmax = no effect / unchanged - Km = increases = decreased substrate affinity - LB plot = only the steepness of slope increases Noncompetitive inhibition (reversible) - Binds at site distinct from subtract active site (able to bind on both E and ES complex) ○ Able to inactive enzyme - Vmax = decreases - Km = unchanged - LB plot = steepness of slope increases + higher y-int Irreversible inhibitor - Binds strongly to active site ○ Usually by formation of covalent bonds with functional groups - Suicide inactivator ○ Def: irreversible inhibitor that can normally perform first few steps but cannot be converted into product → permanently inactive enzyme 4.5 Regulation of enzymatic reactions Allosteric regulation - Allosteric enzymes: usually larger and have 2≤ subunits (catalytic vs regulatory) ○ Have interactions with inhibitors and activators ○ Small metabolites or cofactors eg ATP, CTP - Undergoes conformational change in response to binding of modulator ○ ≠ noncompetitive inhibitors (not necessarily have conformational changes) ○ Modulator: can be inhibitory or stimulatory in nature - eg Aspartate transcarbamoylase ○ CTP formed as end product which inhibits the reaction ○ 2 catalytic trimers + 3 regulatory dimers → changes the quaternary structure ○ ATP as +ve regulator ; CTP as -ve regulator Regulation by reversible covalent modification - eg removal and addition of phosphoryl groups ○ Affects structure and catalytic activity of enzymes ○ Kinase (addition) ; phosphatase (removal) Regulation by proteolytic cleavage of enzyme precursor - eg digestive enzymes are synthesised and stored as inactive precursors ○ aka zymogens / proenzymes EXAMPLE: acute pancreatitis - Caused by activation of pancreatic enzymes within ductile system of pancreas (abnormal regulation) ○ Activated enzymes with not damage proteins on cell surface along blood vessels ○ Serum in blood contains natural inhibitors for trypsin (antitrypsin) - Normal state: pancreatic enzymes should stay inactive until it reaches the duodenum 4.6 Enzymes in health and diseases 1. Drug production - Penicillin (antibiotics): inhibits transpeptidase which is required for the cell wall biosynthesis in bacteria ○ Irreversible inhibitor: makes bacteria lyse → unable to survive - HIV protease inhibitor 2. Lethal nature of irreversible enzyme inhibition 3. For medical diagnosis and treatment