BSCI330 Exam II Review Proteins Covalent modifications to proteins o 1 Phosphorylation Addition of a negatively charged phosphate group to the R group of serine threonine tyrosine b c these all contain OH Phosphate comes from ATP and forms the amino acid residue ADP Reaction is catalyzed by protein kinases enzymes Drives changes in protein structure and activity Because each phosphate group adds two negative charges to the protein which participate in new ionic bonds with neighboring positively charged amino acid R groups or with ions in solution Also because the added phosphate may create a new recognition site that allows other proteins to bind to the phosphorylated protein Removal of the phosphate group dephosphorylation is catalyzed by a second enzyme a protein phosphatase Phosphorylation of a protein by a protein kinase can either increase or decrease the protein s activity depending on the site of phosphorylation and the structure of the protein o 2 Glycosylation Addition of sugars Carbohydrate chains can be O linked to the OH group of serine or N linked to the NH2 group of asparagine o 3 Addition of lipids or glycolipids Addition of phosopholipids and fatty acids to cysteine or N terminal glycine residues to form lipoproteins The fatty acid chain can insert into the hydrophobic core of membranes anchoring a protein to the membrane Enzymes As a catalyst an enzyme does not change G for a reaction Are only required in small amounts Must be unchanged at the end of reaction so that it can cycle back to bind more substrate 1 BSCI330 Exam II Review Catalyzes equally the forward and reverse reactions Can increase the rate of a reaction by 108 to 1012 fold Enzymes lower the EA for a reaction Classification o Oxy reductases catalyze the transfer of electrons from the electron donor to the electron acceptor o Transferases transfer functional groups from one molecule to another o Hydrolases catalyze hydrolysis of chemical bonds o Lyases catalyze alteration or removal of a functional group o Isomerases convert a molecule from one isomer to another o Ligases catalyze joining of two molecules together o Kinases transfer phosphate groups Substrate binding o Enzymes weakly bind a substrate to reversibly form an enzyme substrate complex o This then can react on the enzyme to form a product EP o The product dissociates from the complex into enzyme and product o When enzyme and substrate are mixed in vitro this reaction very rapidly reaches a steady state in which ES is stable and product is produced at a fixed rate o The initial rate of product formation is therefore determined by the amount of ES formed Michaelis Menten Kinetics o As amount of substrate increases the rate increases o Km Michaelis Consttant Generally lies within its substrate s natural concentration range b c synthesizing under used enzymes takes energy 50 point Approx equal to the dissociation constant for enzyme substrate complex Inversely related to the protein affinity Low Km the enzyme has a high affinity for its substrate 2 BSCI330 Exam II Review High Km the enzyme has a low affinity for its substrate o Vmax rate of product formation when the enzyme is saturated with substrate Most sensitive to percent change The point at which the rate of production of substrate is most sensitive to substrate concentration highest slope Rate Vmax S Km S o Enzyme Mechanisms due to the participation of an additional substance called a catalyst the increase in the rate of a chemical reaction of two or more reactants o Catalysis o 1 Stabilizing the transition state Transition state intermediate form contained within an enzyme substrate complex between the product and reactant Have higher energy than either the reactants or products before the Enzymes stabilize transition states by lowering their energy thus reaction occurs also lowering EA o Binding a substrate into an enzyme s active site often forces substrates into a new conformation which resembles the reaction s transition state o This induced fit distorts and strains bonds within the substrate and lowers the EA o 2 Changing substrate reactivity a Formation of temporary covalent bonds Formed between R groups in enzyme s active site and substrate Will speed the reaction b Acceptance or donation of electrons and or protons between R groups cofactors and substrate functional groups May weaken some bonds and strengthen others Will speed the reaction c Ionic interaction Repulsion or attraction of charged R groups with charged functional groups on the substrate May weaken some bonds 3 BSCI330 Exam II Review Prosthetic groups non protein molecules that aid catalysis Will speed the reaction o In some cases these are covalently bound to the enzyme o E g Organo metal compounds in which the metal can donate accept electrons to from the substrate o E g Chromophores small organic molecules which absorb light Cofactors and coenzymes small organic molecules can function as aids in catalysis or binding of the substrate o Many are produced from vitamins which are needed to make other small molecules which are essential components for proteins Multi enzymes o Have more than one active site connected by a tunnel o Allow for efficient transfer of products between active sites o Active site 1 produces ammonia diffuses through the tunnel to active site 2 o Combines w carboxy phosphate to form carbamate highly unstable intermediate o This then diffuses through the tunnel to active site 3 where it is phosphorylated by ATP to produce the final product carbamoyl phosphate Enzyme inhibitors o Reversible inhibitors common in nature Competitive Reversibly bind to active site and compete with substrate Can be displaced by high substrate concentration Do not reduce Vmax Increases Km lower affinity Non competitive Reversibly bind away from active site to cause change in enzyme structure that lowers catalytic efficiency Lower Vmax Do not increase or decrease Km Do not affect substrate binding 4 BSCI330 Exam II Review o Allosteric Inhibition Non competitive inhibition Seen in multi subunit enzymes Made of catalytic subunits and a regulatory subunit An allosteric inhibitor small molecule binds to a site on the regulatory subunit Results in a change in the regulatory subunit s conformation which is coupled to a change in the catalytic subunit s concentration reducing catalytic efficiency o Phosphorylation of enzymes at specific sites and by specific kinases can also stimulate or inhibit their activity Allows one
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