Lecture 4 Biol111 2024 Enzyme Catalysis PDF

3/22/24 What you should know at the end of this lecture That the active site of an enzyme is where a substrate binds Enzymes can change shape when a substrate enters the active site – induc...

3/22/24 What you should know at the end of this lecture That the active site of an enzyme is where a substrate binds Enzymes can change shape when a substrate enters the active site – induced fit The various ways in which an enzyme lowers EA That enzymes have an optimal pH and temperature What irreversible, reversible, competitive and non- competitive inhibitors are The types of regulation – genetic, allosteric, covalent What feedback inhibition is 1 Enzymes lower the activation energy of both exergonic and endergonic reactions 2 1  3/22/24 Active site Typically a close shape match between the active site and substrate (lock and key) – specificity But shape of active site can change when the substrate enters the active site – induced fit 3 Active site 4 2  3/22/24 Active site - hexokinase active site – cleft crevice where catalysis takes place 5 Active site - hexokinase Shape change as the substrate enters – induced fit 6 3  3/22/24 Active site - hexokinase Forms the enzyme substrate complex 7 General strategies of enzyme catalysis (D) Enzyme may form covalent bonds with the substrate itself 8 4  3/22/24 Reaction cycle - lysozyme For a specific example we’ll look at lysozyme Present in your tears Breaks down oligosaccharides in bacterial cell walls 9 What happens at the active site of lysozyme 10 5  3/22/24 What happens at the active site of lysozyme 11 Temperature and pH affect reaction rates Temperatures below optimum – fewer collisions Temperature above optimum – enzyme denatures At pH’s above or below the optimum amino acid residues may be protonated/deprotonated – decreases their activity 12 6  3/22/24 Cofactors Required for catalysis in some enzymes Inorganic ions – Fe, Zn, Cu Complex organic molecules (coenzymes) – Vitamins, NAD+, FAD https://www.google.com/search?q=enzymes&client=safari&rls=en&so urce=lnms&tbm=vid&sa=X&ved=2ahUKEwjD45nS1v_9AhUJzqACH TBFCuUQ_AUoBHoECAEQBg&biw=1324&bih=974 - fpstate=ive&vld=cid:5fefef1d,vid:yk14dOOvwMk 13 Enzyme inhibition Inhibitors may be drugs or toxins or normal metabolites (and hence can regulate activity) Irreversible inhibitors (bind covalently – typically to a serine residue) Reversible inhibitors Competitive – Bind to active site Non-competitive – Binds away from active site 14 7  3/22/24 Irreversible inhibitor - penicillin Penicillin Fleming 1928 Mould Penicillium notatum Inhibits transpeptidase Cross-links in bacterial cell wall 15 Reversible inhibitor - viagra Viagra - Sildenafil Reversible inhibitor of cGMP specific phosphodiesterase type 5 – Breaks down cGMP Causes reorientation of pollen tube growth Ana Margarida Prado et al. Development 2004;131:2707- 2714 16 8  3/22/24 Enzyme control Control how active the enzymes actually are – Allosteric control (fast) (< sec) – Covalent control (relatively fast) (< sec) Control whether enzyme are there or not – Genetic control (slower) (seconds – minutes) 17 Allosteric regulation Molecule (metabolite) binds away from active site – Can stabilise active form (allosteric activator) – switch a metabolic pathway on – Can stabilise inactive form (allosteric inhibitor) – switch a metabolic pathway off Inhibitor often is the end product of a metabolic pathway (feedback inhibition - see next slide) 18 9  3/22/24 Feedback inhibition The end product of a metabolic pathway inhibits an enzyme that catalyses an earlier reaction in the pathway 19 Feedback inhibition and genetic control In addition to feedback inhibition of an enzyme, the end product of a metabolic pathway can inhibit the expression of enzyme that catalyse reactions in a metabolic pathway In the example shown tryptophan inhibits the trp operon as well as enzyme 1 This involves repressor proteins 20 10  3/22/24 Feedback inhibition and genetic control Chapter 16 21 Covalent control Proteins can be phosphorylated and dephosphorylated. Proteins called kinases transfer P from ATP. Other proteins called phosphatases remove P. Phosphorylation can activate some enzymes and deactivate others. Fig 3.64 Alberts et al 2008 Molecular Biology of the Cell 22 11  3/22/24 Enzyme control - an example in frogs Glycogen phosphorylase (enzyme) Found in liver and muscle (slightly different structure - isozymes) Releases glucose from glycogen Complex regulation Both allosteric and covalent control Covalent control involves the phosphorylation via a protein kinase As wood frogs begin to freeze in winter, they release glucose (by increasing glycogen phosphorylase activity), which along with urea – acts as an antifreeze https://www.youtube.com/watch?v=pLPeehsXAr4 23 Enzyme control - an example in frogs In this study the authors compared wood frogs from Alaska and Ohio. Those from Alaska were better at surviving colder temperatures Why? To answer this they looked at kinase (PKA) and glycogen phosphorylase activity in Alaskan and Ohio frogs 24 12  3/22/24 Enzyme control - an example in frogs In Fig 3 Alaskan frogs have more PKA activity (open symbols) compared to Ohio frogs This leads to more glycogen phosphorylase activity (Fig 5) More glycogen phosphorylase activity will lead to more glucose released from the liver and the frogs are able to survive the colder temps in Alaska 25 13