Enzymology PDF

08/10/23 Making life possible: enzymology Learning objective: de ne an enzyme and understand its role in catalysis Learning objective: de ne principles of enzymology Learning objective: illustrate factors that affect enzyme activity. Learning objective: recognise the importance of feedback regulatio...

08/10/23 Making life possible: enzymology Learning objective: de ne an enzyme and understand its role in catalysis Learning objective: de ne principles of enzymology Learning objective: illustrate factors that affect enzyme activity. Learning objective: recognise the importance of feedback regulation, allosteric regulation and cooperativity. Enzymes and their action: De nition: a macromolecule that catalyses a biochemical reaction. Not just proteins ( ribosome are RNA molecules that catalyse peptide bond formation ) Enzyme bioenergetics: Enzymes reduce the activation energy of a reaction. Many covalent and non covalent interactions stabilise 3D structure. These include: 1. Hydrogen bond 2. Hydrophobic interaction 3. Disulphide bridges 4. Ionic salt’ interactions Phosphate-metal interaction Structural speci city: chirality A chiral carbon is one that is attached to 4 different atoms/groups. Stereoisomers: same molecular and structural formula but different spatial arrangement. Enantiomers ( optical isomers ): are stereoisomers that are mirror images- rotate plane of polarised light in opposite ways. A note on naming stereoisomers: Enzyme activity: Reaction rate (V) as a function of substrate (S) concentration. Two parameters: Vmax= maximal rate. Km= [S] for half maximal rate. Effects of pH on enzyme activity: This is dependent on the effect of pH on protein structure. The pH effects the charges on the amino scuds and hence their interactions. KN Acidic form: low pH Neutral form: zwitterion HN Basic form: high pH H N FOH C H CHEAH Foi C H CHEAH poi C H Chaco Effects of temperature: Higher temp= greater kinetic energy of reactants. Vibrations alter interactions hence tertiary structure changes, the enzyme is said to be denatured. Example: glucose metabolism Glucose oxidation: overall reaction. Collie061st 602cg 602191 ADD Pi ATP t 64201g Glycolysis: Only think about stuff in red boxes for now. Metabolic regulation is complex, but key regulatory steps are catalysed by hexokinase (HK) and phosphofructokinase (PFK). Pyruvate has a lower gibbs free energy than glucose and that’s why the reaction proceeds. There are big drops in gibbs when phosphorylation occurs. PFK: a key regulatory enzyme Relatively far from thermodynamic equilibrium Responsive to substrate concentration, not saturated Important allosteric effectors A site of feedback control It catalyses: Fructose 6-phosphate + ATP Fructose 1,6-bisphosphate + ADP + H+ Allosteric regulation: Allosteric regulation is the regulation of an enzyme by binding an effector molecule at a site other than the active site, allosteric site’. Allosteric regulation of PFK: the allosteric effectors: ATP low pH Citrate concentration Basic negative feedback mechanism: PFK Adenine monophosphate q PFK is stimulated by a fall in [ATP] and a rise in [AMP] This increases ATP production Acidi cation accompanying lactate production limits this Enzyme inhibition: Competitive inhibitors: Competitive with substrate at active site of free enzyme. Raises Km, no effect on Vmax because it can be competed out by substrate Non-covalent, reversible Non-competitive inhibitors: Alter enzyme activity by binding to allosteric site Lower Vmax, no effect on Km May be reversible Uncompetitive inhibitors: Bind to enzyme-substrate complex Decrease Km and Vmax. These will be talked about more at a later date Inhibitors in context: acetylcholine esterase in neurotransmission Acetylcholine esterase ( AChE ) inhibitors increase synaptic ACh concentration and are used to enhance neurotransmission e.g in Alzheimer’s. Because of potential toxicity, competitive inhibitors ( which can be titrated ) are preferable to noncompetitive inhibitors. Cooperativity: Steep sections of sigmoid Oxygen dissociation cur ve mean big change is oxygen content for small changes in partial pressure of oxygen. Allostery can produce sigmoid enzyme kinetics: Steep sections of sigmoid curve mean big change in enzyme activity for a small increase in substrate concentration.