Enzymes Chapter 6 Biochem 4511 Figures Essentials of Biochemistry 3rd Ed OSU Custom Edition Principles of Biochemistry 5th Ed Moran et al Lehninger Principles of Biochemistry 5th Ed Nelson Cox Fundamentals of Biochemistry 2nd Ed Voet Voet Pratt Enzymes A very brief history Early studies on enzymes focused on the use of enzymes in food preparation and digestion Yeast fermentation was extensively studied Enzymes A very brief history Rennin or Rennet Some early canteens were made from animal stomachs When milk was stored in these canteens it clumped into cheese Treatment of stomach linings with salt solutions extracted the ability to convert milk to cheese Alcohol fermentation It is possible to convert glucose to ethanol via some action dependent on yeast Yeast ferments The word enzyme comes from the Greek term in yeast reflecting that some molecular component of the yeast NOT the yeast itself is responsible for fermentation Enzymes Nature s Catalysts Catalyst A substance usually in small amounts relative to the reactants that modifies typically increases the rate of a reaction without being consumed Enzymes are protein catalysts that exhibit 1 Higher reaction rates typically 106 to 1012 times faster than the uncatalyzed reactions 2 Milder reaction conditions Below 100 C normal atmospheric pressure and usually near neutral pH 3 Greater reaction specificity 4 The activity is regulated by concentration of substrates products or outside processes allosteric control covalent modification variation of enzyme amount Ribozymes are RNA catalysts Enzymes Vocabulary Words Active site the part of the enzyme which binds the substrate and contains the residues that directly participate in making and breaking bonds Substrate a reactant acted on by the enzyme Inhibitor substance that reduces the activity of an enzyme Cofactor coenzyme small molecule required for some enzyme activity Holoenzyme active enzyme bound to any required cofactors Apoenzyme the enzyme without required cofactor Turnover One step through a reaction cycle Enzymes Carry Out Reactions Other classes are subsets Kinases are transferases for a phosphate group Proteases are hydrolases for amide bonds in proteins Enzymes Speed Up Reactions Don t memorize these but note carbonic anhydrase blood buffering system and chymotrypsin discussed later How well would blood buffering system function without carbonic anhydrase Thermodynamics vs Kinetics G H T S Thermodynamics is the relationship of reactants to products If favorable Greaction a reaction will occur but only kinetics tells you how quickly Activation Energy and Reaction Coordinates Free Energy of Activation Activation Energy In this figure the transition state is the energetically unfavorable high energy complex X The transition state is not very stable therefore the amount of transition state in the reaction mixture is very small Transition state will quickly decompose back to reactants or forward to products Activation Energy and Reaction Coordinates Book figure What can you say about the thermodynamic relationship of A B and B C Why is this diagram best for illustrating G but not a typical reaction Reaction Kinetics and Rate The rate determining step of the overall reaction is the slowest step typically the formation of the transition state Mathematically reaction rate is proportional to e G RT The larger the value of G the slower the reaction rate Rate Determining Step In a multistep reaction the step with the largest activation energy G is the rate limiting step Two different reaction coordinates for conversion of A to P For blue curve first step is rate determining A is larger For red second step is rate determining I is larger Remember this when we reach covalent catalysis Accelerating Reactions Mathematically reaction rate is proportional to e G RT Reaction rate can be increased by temperature T or by increasing the concentration of reactants encounter rate OR Enzymes as Catalysts Enzymes reduce G and therefore speed up reactions Enzymes provide a reaction pathway with a lower activation energy in order to reach equilibrium more quickly Note This is a simple sketch for how catalysts accelerate reactions and is NOT a reaction coordinate for an enzyme catalyzed reaction Classes of Rate Enhancement Exhibited by Enzymes Enzymes increase reaction rates by several methods Most enzymes use one or more of the following 1 Proximity orientation and entropy reduction 2 Preferential binding of the transition state complex transition state stabilization 3 Acid base catalysis 4 Covalent catalysis 5 Metal ion catalysis Proximity Orientation and Entropy Reduction Enzyme catalyzed reactions use standard organic mechanisms but much more efficiently than in solution Proximity local concentration and orientation proper alignment are two obvious physical properties that can be manipulated by an enzyme Bringing reactants close together and aligning them for reaction speeds up reaction Entropy reduction Enzyme Substrate Complex Details of enzyme reaction mechanisms vary but the first step is the formation of the enzyme substrate complex ES complex Note reaction coordinate Substrate undergoes reaction while bound to the enzyme Reaction schemes Uncatalyzed S P Enzyme catalyzed E S ES EP E P complex complex Enzyme Substrate Complex E S ES EP E P complex complex E S ES EP E P Binding equilibria Driven by free energy affinity inherent Energy barrier is solvation desolvation and changes in structure association induced fit Affected by S and P environment sensitive We will revisit this during kinetics Specificity Lock Key vs Induced Fit Lock and Key Hypothesis Induced Fit Hypothesis Active site of enzyme is a perfect match for the substrate substrate binds in conformation for reaction Modest entropy reduction in substrate Substrate and enzyme induce subtle changes to find favorable bound structure for reaction Entropy reduction in enzyme active site and in substrate Specificity in Enzyme Substrate Interactions Enzymes vary in their ability to distinguish between substrates Some enzyme substrate interactions are very specific Enzyme accelerates reaction with ONLY one substrate Most enzymes can act on a range of similar substrates Alcohol dehydrogenase is an enzyme that oxidizes small alcohols including ethanol methanol and isopropanol Although it binds to each reactivity with ethanol is faster than methanol or isopropanol Proximity Orientation 1 Proximity Enzymes bring substrates into contact with