Enzyme mechanism and inhibtion.docx

Enzyme-substrate complex Enzymes are much bigger than their substrates. Different amino acid residues come together at active site in the correct orientation for them to bind to substrate. Active sites often have clefts that exclude water. Most enzymes catalyse only one type of reaction but isn’t al...

Enzyme-substrate complex Enzymes are much bigger than their substrates. Different amino acid residues come together at active site in the correct orientation for them to bind to substrate. Active sites often have clefts that exclude water. Most enzymes catalyse only one type of reaction but isn’t always true- some peptidases can also function as esterases. Lock and key model States that enzyme active site is exactly the correct shape for substrate to bind to. Problem: small molecules that have a similar shape and part of the substrate cannot bind. Induced fit model. Substrate induces a change in confirmation so it can fit into the active site of the enzyme. Substrate specificity- stereospecificity. Only binds to one optical isomer as the residues are in specific order and need to interact with the active site. Rate enhancement Enzyme can only catalyse reactions which occurs spontaneously in nature. Rate enhancement is obtained from ratio of catalysed to un-catalysed rate constant Kcat/Kun Reactions proceed via transition state. Every reaction goes via transition state. Need to put energy into the system to reach the transition state before forming products. Transition state is the point of highest energy on the progress of a reaction curve. It is where bond breakage and formation occur. Enzyme speeds up reaction because they reduce the activation energy that’s needed. *The main way enzymes reduce the activation energy is by stabilising the transition state. * With induced fit model the best fit for all Is between enzyme and the transition state molecule. //// Irreversible inhibitors vs reversible Irreversible inhibitor never comes off once it binds to the enzyme due to covalent bonds. Reversible inhibitor binds and comes off because it doesn’t form covalent interactions. How to tell apart between reversible and irreversible? Dialysis- has a semi-permeable membrane which allows small molecules to cross only. Irreversible If irreversible inhibitor is mixed with enzyme and placed into a dialysis tube and put in a buffer. Nothing happens due to the inhibitor binding to enzyme and forming covalent interactions forming a big complex that is too large to leave the semi-permeable membrane of the dialysis tube. Reversible If reversible inhibitor is mixed with enzyme and placed into a dialysis tube and put in a buffer. Inhibitor moves out due to it not forming any covalent interactions with the enzyme and it is still enough to move out of the dialysis tube if the concentration gradient is big enough as the inhibitor is small to enough to move out. Examples Sarin is an example of irreversible inhibitor of acetylcholinesterase. Acetylcholinesterase catalysed the breakdown of acetylcholine. This is vital as neurotransmitter needs to be broken down. Acetylcholinesterase has a very reactive serine OH side chain at the active site. Water then hydrolyses to regenerate the OH at the active site. Sarin also reacts with the serine residue in the active site however the complex formed is highly stable and can’t be hydrolysed by water, so the enzyme is permanently inactivated. Suicide inhibitors – subgroup of irreversible inhibitors. Simple reactive molecules lack specificity as enzyme inhibitors, so a molecule is made that the enzyme thinks to be as the substrate and processes it. The molecule becomes irreversibly bound and inactivates the enzyme. The molecule itself is inert until it binds to actives site and becomes irreversible. Eg- 5-fluorouracil= anticancer drug Process of converting uracil to thymidine Uracil goes through several steps of metabolism to generate deoxyribose monophosphate. Deoxyribose monophosphate is the substrate for thymidylate synthase. It forms covalent bond between thymidylate synthase enzyme and deoxyribose monophosphate. Then a small molecule called tetrahydrofolate binds to deoxyribose monophosphate and donates its CH2. The previously 2 bonds made are broken and thymidine is released. When the same process happens with 5-fluorouracil, when it forms the deoxyribose monophosphate with tetrahydrofolate and the covalent bond between thymidylate synthase enzyme and deoxyribose monophosphate. This becomes very stable molecule and doesn’t break down. Thymidine cannot be produced, and DNA replication can’t happen. Types of reversible inhibitors Competitive inhibitor- binds same site as the substrate. Uncompetitive inhibitor Non- Competitive inhibiter- binds elsewhere on the enzyme and not the active site. Competitive inhibitor Binds same site as substrate and blocks access for the substrate. When concentration of substrate is low, presence of the inhibitor is dramatic as it blocks the active site. When concentration of substrate increases, it’s possible for the substrate to outcompete the inhibitor and bind to the active site. Enzyme cannot bind both the substrate and the inhibitor at once. Completive inhibitors DON’T EFFECT the Vmax as high levels of substrate can outcompete the inhibitor. Ki= dissociation constant of the enzyme-inhibitor complex. It is a measure of affinity between enzyme and inhibitor. Smaller the Ki, the tighter it binds as higher the affinity. Km is effected by competitive inhibitors. Non-Competitive inhibitor Bind elsewhere on enzyme, induces confirmation change and prevent catalysis. It effects the Vmax as it cannot be outcompeted with increasing substrate concentration. Km isn’t effected by non-competitive inhibitors. Lineweaver-burk plot can be used to distinguish types of inhibitors. Plot 1/[S] against 1/[V] Produces a straight line. Point it crosses the Y-axis is = 1/ Vmax Presence of Competitive inhibitor Crosses the y-axis at the same point as it DOESN’T affect the Vmax. Presence of non-competitive inhibitor Crosses the y-axis at a different point as it DOES affect the Vmax. Example Methotrexate is a competitive inhibitor for the enzyme dihydrofolate reductase.