ACJC Enzyme catalysis lecture notes

H3 Chemistry 9813 Enzyme catalysis Anglo-Chinese Junior College Class of H3 Chemistry ‘26 Enzyme Catalysis lecture notes Prepared by: Colin, Chloe, Zheng Yang (H3 Class ‘26) Special thanks to H3 chemistry teachers Section Content Page 1 Intro to enzymes ● Induced fit model 2 3 2 Types of enzyme interactions ● Acid-Base (proton acceptor/donator) ● Electrostatic Catalysis ● Metal ion catalysis ● Bond strain ● Covalent catalysis 4 5 6 8 9 3 Transition state theory ● Erying equation 11 4 Enzyme kinetics ● The Michaelis constant 𝐾𝑚 ● Michaelis-Menten equation and graph 12 13 5 Enzyme regulation & inhibition ● Reversible inhibition ● Irreversible inhibition 14 16 6 Annex ● A-Derivation of Michaelis-Menten equation 17 1

H3 Chemistry 9813 Enzyme catalysis Intro to enzymes In catalysis, enzymes act as specific biological catalysts in the breakdown or assimilation of substances, speeding up the rate of metabolic reactions. Nature of enzymes: ● Most enzymes are globular (looks round) proteins, consisting of one or more polypeptides coiled and folded together to form a globular structure. ● Enzymes are organic catalysts produced by living cells. They remain unchanged at the end of a reaction and do not alter the nature or properties of the end-product(s) of the reaction. ● Enzymes are highly selective in their action as the active site has a specific 3D confirmation that complements only targets specific functional groups ( group specificity ). ● Rates of enzymatic reactions are affected by pH, temperature, enzyme concentration and substrate concentration. ● Enzymes have an active site , which is the catalytic centre of the enzyme, where the substrate binds for catalysis to take place. Common Features of Active Site: 1. The active site is a three-dimensional cleft formed by R (variable functional) groups of amino acids of the polypeptide ○ The active site has a specific 3D conformation that is complementary to the substrate, which accounts for the specificity in the types of substrate that it can act on. ○ The specificity of binding depends on the arrangement of amino acids in an active site. 2. The active site takes up a relatively small part of the total volume of an enzyme. ○ Typically consist of 3 – 12 amino acids. ○ These amino acids may be found far apart along the polypeptide chain, but were brought near together through folding. 3. Substrates bind to enzymes at the active site by multiple weak interactions such as Van der Waals forces and hydrogen bonds . 2

H3 Chemistry 9813 Enzyme catalysis Substrate Binding Mechanisms Originally, enzymes were thought to be ‘locks’, through which a substrate ‘key’ could be fitted. The lock would remain unchanged after each reaction, and they would have to already be conformationally complementary for the interaction to occur. However, more recently, the induced fit model was proposed, which states that enzymes are more like a mould which wraps itself around the substrate, and adjusts its shape accordingly. Today, the ‘lock and key’ model and ‘induced fit model’ are thought to work in conjunction, as two separate mechanisms of enzyme binding. Mechanisms of substrate binding Different enzymes favour different modes of binding, depending on their substrate or function. These modes include: ● Uniform binding (‘lock and key’), where the active site has a high affinity for binding for both the substrate (S) and the substrate in transition state (S ‡ ). ○ This means that the enzyme naturally binds to the substrate by default without needing to change much in conformation. ○ This lowers the energy levels of both the enzyme-substrate complex (ES) and enzyme-substrate transition complex (ES ‡ ). ● Differential binding (‘induced fit’), where the active site has a high affinity for just the substrate in transition state. ○ This means that while the substrate and enzyme are not a good ‘fit’ initially, the enzyme shifts its conformation such that it fits well with the transition state. ○ This lowers the energy level of ES ‡ whilst the energy level of ES remains high. Enzymes that are usually saturated favour differential binding. When an enzyme is saturated, all its active sites are occupied by substrate molecules, forming ES(es) which are stable. Thus, the enzyme must overcome the activation energy (E a ) to form ES ‡ for catalytic action. Saturated enzymes want to be able to favour the formation of ES ‡ instead of retaining ES. Thus, they favour differential binding. However, some substrates are able to bind to enzymes through either mechanism. Specifically, these substrates must be small and unbound . Simple, small substrate molecules do not require large conformational change in the enzyme to bind, and can also be held within the active site with just a few bonds. Unbound substrates (i.e. substrates 3

H3 Chemistry 9813 Enzyme catalysis which are low in concentration) go through uniform binding due to the enzymes’ active sites being largely unsaturated. 4

H3 Chemistry 9813 Enzyme catalysis Types of enzyme interactions I. Acid-Base Catalysis Amino acids in the active site can act as acids and bases, to stabilise charges that appear in the substrate’s transition state. For example, histidine has a imidazole (highlighted) side chain which has a pKa of around 6.0, which is close to physiological pH, especially that of the blood, at 7.4. This means that there is a sizable amount of conjugate acid present even at physiological pH, and thus it can act as a kind of buffer. This allows enzymes to protonate and deprotonate substrate molecules in various steps. The two carboxylic acid side chains in aspartic and glutamic acid have pKa values of around 4.0, but these can be higher in reality when found in the 3D confirmation of an enzyme. Hence they can be protonated and serve as acids. In an actual enzyme, acid-base looks as in the example of Ribonuclease A, which hydrolyses an RNA molecule (a polymer of smaller molecules called ribonucleotides): 5

H3 Chemistry 9813 Enzyme catalysis II. Electrostatic Catalysis Typically, charges during catalysis can be present for reasons such as: ● Substrate molecules being charged; ● Transition states being charged (deprotonation, bond fission etc.). Enzymes catalyse reactions involving charges by: ● Surrounding substrate molecules with like charges, therefore destabilising them and driving the reaction towards forming the transiti