Enzymes Mechanism and Catalysis Enzymes DO NOT change the equilibrium constant of a reaction Enzymes DO NOT alter the amount of energy consumed or liberated in the reaction standard free energy change G Enzymes DO increase the rate of reactions that are otherwise possible Enzymes DO decrease the activation energy of a reaction G Enzymes DO increase the rate of reactions that are otherwise possible Enzymes DO decrease the activation energy of a reaction G The classic way that an enzyme increases the rate of a bimolecular reaction is to use binding energy to simply bring the two reactants in close proximity In order for a reaction to take place between two molecules the molecules must first find each other This is why the rate of a reaction is dependent upon the concentrations of the reactants since there is a higher probability that two molecules will collide at high concentrations The enzyme organizes the reaction at the active site thereby reducing the cost in terms of ENTROPY How do enzymes catalyze biochemical reactions involves basic principles of organic chemistry What functional groups can be involved in catalysis almost all alpha amino and carboxyl groups are tied up in peptide bonds R groups are involved in catalysis asp glu his lys ser cys tyr catalysis occurs when substrate is immobilized near these residues at the active site General Acid Base Catalysis General acid base catalysis is involved in a majority of enzymatic reactions General acid base catalysis needs to be distinguished from specific acid base catalysis In General acid base catalysis the buffer aids in stabilizing the transition state via donation or removal of a proton Therefore the rate of the reaction is dependent on the buffer concentration as well as the appropriate protonation state General Base Catalysis I Ester Hydrolysis General Base Catalysis II Ester Hydrolysis The hydrolysis of esters proceeds readily under in the presence of hydroxide It is base catalyzed However the rate of hydrolysis is also dependent on imidazole buffer concentration Imidazole can accept a proton from water in the transiton state in order to generate the better nucleophile hydroxide It can also re donate the proton to the paranitrophenylacetate in order to generate a good leaving group General Acid Catalysis Ester Hydrolysis Electrostatic interactions are much stronger in organic solvents than in water due to the dielectric constant of the medium The interior of enzymes have dielectric constants that are similar to hexane or chloroform Catalysis by Metal Ions Catalysis I Ester Hydrolysis Metal ions that are bound to the protein prosthetic groups or cofactors can also aid in catalysis In this case Zinc is acting as a Lewis acid It coordinates to the non bonding electrons of the carbonyl inducing charge separation and making the carbon more electrophilic or more susceptible to nucleophilic attack Catalysis by Metal Ions Catalysis II Ester Hydrolysis Metal ions can also function to make potential nucleophiles such as water more nucleophilic For example the pka of water drops from 15 7 to 6 7 when it is coordinated to Zinc or Cobalt The hydroxide ion is 4 orders of magnitude more nucleophilic than is water Covalent Catalysis Acetoacetate Decarboxylase O H 3C Lys NH3 H 3C O CH 3 O C O H 2C O hydrolysis O CH3 Lys N H CH3 B H 2O CH 3 Lys N H CH 2 CH 3 O Lys O CO2 N H CH 2 BH Enzymes physically interact with their substrates to effect catalysis E S ES ES EP E P where E enzyme S substrate ES enzyme substrate complex ES enzyme transition state complex P product EP enzyme product complex Enzyme and substrate combine to form a complex Complex goes through a transition state ES bound substance is neither substrate nor product A complex of the enzyme and the product is formed The enzyme and product separate All of these steps are governed by equilibria Substrates bind to the enzyme s active site pocket in the enzyme Substrates bind in active site by hydrogen bonding hydrophobic interactions ionic interactions QuickTime and a Video decompressor are needed to see this picture QuickTime and a Video decompressor are needed to see this picture QuickTime and a Video decompressor are needed to see this picture QuickTime and a Animation decompressor are needed to see this picture Enzyme Substrate Interactions Lock and key model substrate key fits into a perfectly shaped space in the enzyme lock Induced fit model substrate fits into a space in the enzyme causing the enzyme to change conformation change in protein conformation leads to an exact fit of substrate with enzyme Following catalysis the product s no longer fits the active site and is released Enzymes and Enzyme Kinetics Strain and Distortion model The binding of the substrate results in the distortion of the substrate in a way that makes the chemical reaction easier Enzyme Kinetics The rate of the reaction catalyzed by enzyme E A B P is defined as A t B t or P Enzyme activity can be assayed in many ways or t disappearance of substrate appearance of product continuous assay end point assay For example you could measure appearance of colored product made from an uncolored substrate appearance of a UV absorbent product made from a non UV absorbent substrate appearance of radioactive product made from radioactive substrate Enzymes and Enzyme Kinetics Higher temperature generally causes more collisions among the molecules and therefore increases the rate of a reaction More collisions increase the likelihood that substrate will collide with the active site of the enzyme thus increasing the rate of an enzyme catalyzed reaction Each enzyme has an optimal pH A change in pH can alter the ionization of the R groups of the amino acids When the charges on the amino acids change hydrogen bonding within the protein molecule change and the molecule changes shape The new shape may not be effective The diagram shows that pepsin functions best in an acid environment This makes sense because pepsin is an enzyme that is normally found in the stomach where the pH is low due to the presence of hydrochloric acid Trypsin is found in the duodenum and therefore its optimum pH is in the neutral range to match the pH of the duodenum At lower concentrations the active sites on most of the enzyme molecules are not filled because there is not much substrate Higher concentrations cause more collisions between the molecules With more molecules and collisions enzymes are more likely to encounter molecules of reactant The