BISC 330L Biochemistry Spring 2015 Professors Dr Lin Chen Tel 213 821 4277 Email linchen usc edu Office RRI 204C Office Hours RRI 204C Tuesday 1 2 pm Dr Fabien Pinaud Tel 213 740 2262 Email pinaud usc edu Office RRI 204A Office Hours RRI 204A Tuesday 1 2 pm Course Director Dr Christa Bancroft Tel 213 740 5553 Email cbancrof usc edu Office ZHS 470 Office Hours ZHS 470 Tuesday Thursday 11 12pm Required Text Berg Tymocko Stryer BIOCHEMISTRY 7th ed 2012 BISC 330L Lab Manual available in USC Bookstore D Benson Berg Tymoczko Stryer Biochemistry Seventh Edition Chapter 8 Enzymes Basic Concepts and Kinetics Sections 8 1 8 2 Basics Copyright 2012 by W H Freeman and Company Concepts Enzymes are powerful catalysts with high substrate specificity DO increase the rate of biochemical reactions but DO NOT alter the equilibrium of a reaction DO NOT affect the energetics of the reaction G Enzymes often require co factors coenzymes or metals for catalytic activity Change in free energy G is a valuable thermodynamic parameter to estimate if a reaction can take place and evaluate the energy cost of the reaction Enzymes Basics Enzymes are generally proteins Sometimes RNA ribozymes Trypsin Group I Ribozyme domain Act as catalysts for biochemical reactions Form complexes by binding a substrate at an active site provide unique environment for catalysis E S ES Binding is HIGHLY SPECIFIC Enzymes are not chemically altered by the reaction Enzymes do not change the equilibrium constant Keq of the reaction Enzymes INCREASE THE RATE of a biochemical reaction by lowering the activation energy to go from substrate S to product P Catalytic power of enzymes Reaction Rate enhancement Note Rate constants symbol is a small k This is not the equilibrium constant symbol big K A few examples Catalytic substrate specificity of enzymes 1 substrate they recognize 2 reactions they catalyze Enzymes are EXTREMELY specific e g Proteases catalyze the hydrolysis of peptide bonds in proteins More than 550 different proteases in the human body Thrombin Proteolytic enzyme involved in blood clotting cascade Recognizes a specific sequence of amino acids Leu Val Pro Arg Gly Ser Very specialized highly specific only where blood need to clot Catalytic substrate specificity of enzymes continued Trypsin Enzyme involved in digestive system Cuts after Lys or Arg Very specialized but less specific than thrombin will cut any protein it encounters PDB 2PTN Catalytic site Cofactors Cofactors are small molecules that enzymes need to execute biochemical reactions apoenzyme cofactor holoenzyme Cofactors are either coenzymes or metals Coenzymes can be weakly bound cosubstrates associate or dissociate during reaction tightly bound to enzymes prosthetic groups Coenzyme cosubstrate NADH Lactate dehydrogenase motif Box 7 2 Princ of Biochem 5th edition 2012 Pearson Metals Zn2 Carbonic anhydrase Understanding how enzymes work energetics Whether a reaction takes place at all and the degree to which enzymes accelerate the reaction depends on energy difference between reactant and products FREE ENERGY G is a measure of useful energy that is energy capable of doing work Difference in FREE ENERGY G is a useful thermodynamic value to estimate if a reaction can take place spontaneously or not G Gproducts final state Greactants initial state Reactants Products If G 0 negative value reaction proceed to the right spontaneously exergonic If G 0 reaction at equilibrium and no net changes If G 0 positive value reaction left to right does not take place spontaneously Note however reaction right to left is spontaneous endergonic reaction Spontaneous reaction release of energy exergonic no need for extra input of energy Non spontaneous reaction need for extra input of energy if you want the reaction to go left to right endergonic Understanding how enzymes work energetics continued Note G tells nothing about the rate of a reaction gives a clue only about the need or not for energy input to drive the reaction in a particular direction L R or R L This clue is important to understand the role of an enzyme in a reaction Consider the reaction A B C D G G RT ln Note these are starting molar concentrations values of reactants and products not Keq Only 3 parameters to consider to determine the actual free energy change of any reaction 1 G Standard free energy change 2 Actual concentrations of reactant and product in solution 3 Temperature T G standard free energy change is a standard value to simplify calculation of G when each reactant and each product are initially at 1 M concentration and for pH 7 0 G Understanding how enzymes work energetics continued G is a reference value for a specific type of reaction at equilibrium G can be determine by measuring conc of reactants and products when the reaction has stopped NO net changes between reactant and products EQUILIBRIUM Note these are molar concentrations values of reactants and products at equilibrium Keq Equilibrium does not mean A B C D Recall at equilibrium G 0 so G and G units Kilojoule kJ Kilocalories kcal G G RT ln 0 G RT ln G RT ln K eq K eq e G RT Direct relationship between G and K eq Remember that criterion for reaction spontaneity is G not G With G 0 reaction can still be spontaneous by adjusting concentration of reactant and products Understanding how enzymes work kinetics Enzymes alter the rate of a biochemical reaction NOT its equilibrium Reaction has reach equilibrium Enzymes accelerate the time it takes to reach equilibrium Enzymes have no effect on the equilibrium itself However there is a relationship between reaction rate and equilibrium constant S P kF kR kF rate constant of forward reaction kR rate constant of reverse reaction Velocity rate of forward reaction VF kF S Velocity rate of reverse reaction VR kR P At equilibrium VF VR so kF S eq kR P eq Keq Review of Concepts Enzymes are powerful catalysts with high substrate specificity DO increase the rate of biochemical reactions but DO NOT alter equilibrium constant Keq DO NOT affect the energetics of the reaction G Enzymes often require co factors coenzymes or metals for catalytic activity Change in free energy G is a valuable thermodynamic parameter to estimate if a reaction can take place and evaluate the energy cost of the reaction Berg Tymoczko Stryer Biochemistry Seventh Edition Chapter 8 Enzymes Basic Concepts and Kinetics Section 8 3 Transition state Copyright 2012 by W H Freeman and Company Concepts Enzymes increase the rate of reactions by