Enzymes.docx

Unit 7: Enzymes - Enzymes do not change equilibrium - they change kinetics. - Binding forces holding an enzyme and substrate together can be ionic, electrostatic, hydrophobic, or some combination. Covalent bonds are less common because they require more energy and release is more difficult. - Potential energy is stored in bonds, enzymes work to reconfigure bonds to get them to the most stable conformation and you get the potential energy out. - Transition state  activation energy between relatively unstable molecule and the more stable. - G  Free energy. - Stability difference is G. Size of the mountain that must be overcome is difference between G and transition state (‡). Backwards hill is starting from the product going to reactant, but typically the hill will be bigger. - Transition state is distorted and doesn’t look like either of the reactant or products. - Catalyst will make the reaction go faster by PROMOTING the transition state, or LOWERING the activation energy. Q: how is this catalyst lowering the energy? o Provide a surface for binding. o Catalyses hydrogen by added onto double bounded carbon by sticking to the surface. (versus floating around in air). o Entropy is reduced, they are not in three dimensions they are in two. Much more likely to find each other. o Promoting bond formation by making things less spread out and by promoting the transition state. - Enzymes are protein catalysts (2/3) (no metal at all, just collections of amino acids). Amino acid residues are brought together and interact with the substrate to directly lower transition state. Have sites that bind and react and provide some kind of surface that catalyzes the reaction. - Sometimes an active site can bind more than one substrate. Because enzymes work by contact, you can narrow down where the active site must be. - EX: Enzyme ribonuclease binds the nucleotide uracil using hydrogen bonds. Specificity: how does it bind? Forms 3 H bonds to uracil. The enzymes ability to bind to a substrate and only that substrate is specificity. - Enzymes can also use large metal atoms (1/3 of enzymes). EX. Cu for cytochrome oxidase. - Metal ions can be held by organic/metallorganics. - Coenzymes: unique structure that helps the enzyme work by making the “active site” more chemically reactive. Most are vitamins; whenever the cofactor is not a metal (NAD) - Cosubstrate: a noncovalently bound coenzyme - Prosthetic group: if the cofactor is covalently bond (literally stuck on) - Holoenzyme: an enzyme with its cofactors bound (whole) - Apoenzyme: an enzyme without its cofactors (empty) Enzyme Classifications: 1. Oxidoreductases: move electrons around (hydride ions or H atoms) (oxidation reduction) 2. Transferases: Group transfer reactions 3. Hydrolases: hydrolysis reactions (transfer functional groups to water) 1 4. Lyases: create double bonds by removing groups. Elimination of functional groups (usually H+ and O- to form a C=C double bond 5. Isomerases: Add isomerase. Transfer groups within molecules to yield isomeric forms. 6. Ligases: Forming bonds by condensation. Requires ATP hydrolysis to provide the energy - EC 1 number: the kind of reaction catalyzed by the enzyme (above) nd st - EC 2 rdmber: (1 subcndss) the atoms or bonds acted on by the enzyme - EC 3 number: (2 subclass) the enzyme type which catalyzes the specific reaction on the specific type of atom or bond - EC 4 number: Serial number for each enzyme in subclass 2. NOTE: type of reaction is what happens, reaction mechanism is how it happens. Catalytic Mechanisms: 1. Acid-base catalysis 2. Covalent catalysis 3. Metal ion catalysis 4. Proximity and orientation effects 5. Preferential binding of transition state complex General Acid-Base Catalysis / Specific Acid-Base Catalysis: - If an enzyme moves protons around, it takes place in acid base catalysis. Proton transfer lowers the free energy of a reaction’s transition state. - Acid form: has a proton on it (donor). Base form: accepts a proton - Side chains of amino acids Asp, Glu, His, Cys, Tyr and Lys have pK’s near physiological pH that allows them to acts as acid/base catalysts. - The catalytic activity is sensitive to pH which influences the protonation of side chains. Ex. RNase A acts as a proton donor and proton acceptor in cleaving RNA for hydrolysis. Covalent Catalysis - Accelerates reaction rates by forming catalyst-substrate covalent bond. - Enzyme has a nucleophile that attacks the substrate, forming a covalent bond with electrophile. - Always two steps. Enzyme must be unchanged, therefore you must break the covalent bond and release the other half of the molecule. - General hydrolysis reaction: Enzyme attacks one of the two sides of the bond. Ex. Acetoacetate to acetone via Schiff base intermediate Metal Ion Catalysis: - Transition metal ions within the active sites of enzymes are strongly bound to the enzyme. Alkali and alkaline earth metal ions are more loosely bound - Metal ions orient a substrate within the active size or neutralize a negative charge on the substrate. - Mg and P are the perfect match because their charges fit together. Mg fits between minuses on the phosphate. - Metals that can undergo redox changes (Mn and Fe ) act as redox catalysts. EX. Carbonic anhydrase uses zinc to hold a water molecule in attack position. o Zinc binds N (three arranged in just a way to bind the Zn enzyme). CO can reac2 with H O 2 Proximity and Substrate Orientation Catalysis: 2 - Important for biomolecular reactions. - 3D nature of active site allows two substrates to be oriented properly and brought into closer proximity than possible in bulk solution  allows it to occur more efficiently than when contact is random. Electrostatic/Ionic Catalysis: - When a charged substrate is guided to the active site by the charge distribution of the enzymes surface amino acids Ex. Anion superoxide interacts with positively charged amino acids on the surface of the enzyme superoxide dismutase. - An electrostatic interaction occurs between substrate and amino acids within the active site. - Chemical reactivity of the substrate is enhanced. Transition State Binding: - The charge distribution on the substrate complements the charge distribution on the enzyme’s active site. - The substrate is distorted until it can fit in the active site. This distorted substrate is the transition state. Ex. Lysozyme cleaving polysaccharides. Theory: - Change G value from G‡(uncat) to G‡(cat) - Conversion step  conversion is turned into product. Catalyze implies that there are three steps occurring. - Enzyme binds to substrate, substrate goes to product (transformation or actual catalyses), enzyme is released from product. - Activation energy for G‡(catalyzed) must be LESS THAN < G‡(uncatalyzed) - Three steps: E + S  ES  EP  E + P - Enzyme does not affect the overall equilibrium. (S  P) Kinetics are what is affected by the enzyme. Does not change HOW the bonds rearrange, rather how quickly they rearrange. - Catalysis only works in the middle, it does not work in the ends. The ground states of reactants and products do not change, only the transition states - Affect RATES not EQUILIBRIUM Rate Equations: - V: velocity of an equation - k: rate constant. tells you the rate, but is NOT the rate itself - First order kinetics: For S  P: V = k[S] o By varying concentrations you determine if it is first order or not - Second order kinetics: For S +1S 2 P: V = k[S1][S2] - G‡ is in k per molar units? you can directly convert the two. Another way of saying the rate constant. (logarithmic relationship) k will change a lot more than G - Rate enhancement: how much the k changes. o k(catalyzed)/k(uncatalyzed) Q: how to enzymes change the transition state? - They bind to the substrate, product and transition state. Will have a certain affinity for both, but also has a certain high affinity for the transition state 3 - Enzymes bind the transition state of the reaction to make it lower in energy  stabilize it and make it more likely to happen (thus reaction more likely to happen). Enzyme Action: - An enzyme that binds it substrate too well never changes the substrate, it ends up being a transport protein. - Something that is too complementary to its substrate will make it harder for the substrate to continue on to the product. - Myoglobin  sticks to oxygen and sits there, doesn’t do anything (will never change the substrate). - Enzyme must be complementary to transition state. Able to bind weakly at substrate, that pulls the substrate to the transition state (and beyond). - EX: DNA binding proteins. A protein that binds DNA but does not cut it  binds as a straight helix in its most stable form. - DNA endonucleases cut DNA after bending it. Cuts the DNA right near the middle of the bend. - Binding the transition state stabilizes it and lowers the G‡ - If you take the differences in k you get numbers around 60-100 kJ/mol needed for normally observed enzymatic rate enhancements. Entropy Reduction: - Enzymes can also work just by bringing things together. - Binding two things in the right orientation. - If you connect the two ends, you get a significant increase in rate. The two molecules can rotate but they are stuck together. Reducing the entropy/chaos. - You are not activating the groups - Reduce entropy/rotation further and the rate increases even more. Make the other ends more “solid” by decreasing potential rotations Enzyme Binding: - Some enzymes move when the substrate binds. The substrate bends the enzyme around itself.  Induced fit - Some enzymes are not flexible and have a structure binding site to match the substrate  lock and key Desolvation: - stripping water away to bind something - Water is around the substrate, and when it binds the surface of the enzyme and substrate must be desolvated before they can bind. - Allows the hydrophobic effect to work. Enzyme Kinetics: - Curve will never follow a straight line, the reaction will eventually even and flatten out. Slope starts off large positive, and then changes to zero. CONSTANTLY CHANGING. - When you mix 1uM substrate with enzyme and time how fast product is produced you get an initial line that starts to curve. - The lower the substrate, the slower the rate of reaction, and the less product you will have at equilibrium. (Equiilibrium changes depend on [S]) 4 Steady State: - In normal enzyme kinetics, the pre-steady state happens too fast to measure