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UCONN / Biology / BIOL 2000 / How do we increase the rate of a reaction?

How do we increase the rate of a reaction?

How do we increase the rate of a reaction?

Description

School: University of Connecticut
Department: Biology
Course: Biochemistry
Term: Fall 2016
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Cost: 50
Name: Biochem Exam 2
Description: These notes cover the notes for Exam 2
Uploaded: 11/10/2016
9 Pages 123 Views 0 Unlocks
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How fast will reaction occur?




■ Why phosphorylate glucose?




■ Enzymes cannot make unfavorable reactions favorable, only increase velocity ■ How do we increase the rate of a reaction?



Bioenergetics  ■ Interactions and reactions lead to energy transformations ■ Critical for all living organisms Reaction Properties  ■ Will it occur at all? ■ Yes=spontaneous, products are energetically favored, depends on free energy  difference between reactants and products ■ EnzymDon't forget about the age old question of Why build telescopes on the ground when we can launch them into space?
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es cannot make unfavorable reactions favorable, only increase velocity ■ How do we increase the rate of a reaction? ■ Energy is always required to convert reactants into products (activation energy) ■ Example reaction: ATP+H2O<-->ADP+Pi+H+ ■ Favorable due to increase in entropy (two species-->3 species) ■ Removal of negative charges from ATP, decrease in interaction between neg  charges ■ Gibbs Free Energy=The amount of energy available to do work ■ Energy given off in ATP hydrolysis, for example, is able to do work ■ Coupled reactions: Pair an unfavorable reaction with a favorable one so that both can  occur ■ Favorable reaction may also use product from unfavorable reaction, driving  equilibrium for unfavorable to the right ■ First Law of Thermodynamics: Energy is neither created nor destroyed, only transferred ■ Organisms are open systems which lose and gain energy from environment ■ Change in energy=heat + work ■ Second Law of Thermodynamics: Systems favor disorder/randomness–entropy ■ Resonance forms lead to increased entropy ■ Reactions want to go to equilibrium and minimize potential energy ■ At equilibrium, forward and reverse reactions occur at the same rate ■ Organisms must shift away from equilibrium, because at equilibrium there is no  free energy to be harvested to do work ■ Favorable reactions release energy and are exergonic ■ Unfavorable reactions absorb energy and are endergonic ■ Example: ATP hydrolysis ■ Free energy of ATP>Free energy of products ■ Entropy of ATP<Entropy of products ■ Favorable reaction conditions, due to high energy bond between negatively  charged phosphate groups ■ Gibbs Free Energy=Energy available to do work■ Difference in chemical bond energy ■ Energy change necessary to reach equilibrium ■ Magnitude of Gibbs free energy tells you how much energy is available and how  far the system is from equilibrium (0 free energy) ■ A large Keq means that almost all reactants are converted to products ■ If you can measure Keq, you can calculate delta G naught ■ Knowing delta G for any reaction tells you how much energy is required or given off by  the reaction ■ Delta G naught prime used for biological systems, same as delta G naught, except  measured at pH 7 ■ Tells you ratio of products to substrate at equilibrium, and gives point of reference ■ Delta G naught prime for ATP hydrolysis is VERY negative due to negative  charge repulsion in phosphate groups ■ Reverse reaction (addition of phosphate to ADP) has same magnitude of delta G,  but opposite sign (positive and unfavorable) ■ Coupled reactions–allow unfavorable reactions to go forward ■ Example TCA Cycle: L-malate+NAD+-->OAA+NADH+H+ has G of 7kJ/mol ■ Acetyl CoA+OAA-->Citrate+CoA+H+ has G of -8kJ/mol ■ Second reaction provides energy for first to occur, also uses product of  first reaction, which drives the equilibrium of the first reaction to the  right–Le Chatelier's principle ■ As long as net of coupled reactions is negative, it will go forward ■ Example: Glucose+Pi-->Glucose-6-Phosphate (+ delta G) ■ ATP-->ADP+Pi (- delta G) ■ Reactions couple to make glucose phosphorylation favorable ■ Why phosphorylate glucose? ■ Trap glucose in cell by addition of negative charge, so it can be used for  energy How fast will reaction occur?  ■ Enzymes do not catalyze unfavorable reactions, only speed up favorable ones ■ Enzymes allow reactions to occur within seconds-->adaptive advantage ■ Function of enzymes determines effectiveness of drugs in different people ■ SNPs affect the efficiency of certain enzymes, drug effectiveness dependant on  how fast it is metabolized ■ Carbonic anhydrase increases rate of addition of water to carbon dioxide by a factor of 8  million ■ Properties of Enzymes■ Specificity ■ Catalysis ■ Regulation–cell only produces what it needs ■ Specificity–enzymes catalyze very specific reactions depending on structure and ability to  form noncovalent interactions ■ Example: Trypsin v. Thrombin→both catalyze peptide bonds, but  recognize different R groups ■ Trypsin cleaves next to Lys or Arg–positive charge binds with negative charge on  Trypsin, which stabilizes the trypsin-protein complex ■ Thrombin only cleaves next to Arg ■ Catalytic site comes together as a result of protein folding ■ The amino acids that make up the active site do not need to be next to each other  in the initial sequence ■ Mutations in catalytic site lead to decreased or eliminated catalysis ■ Enzymes are stereospecific ■ Example: enzyme that interacts with glucose can’t catalyse galactose, because  galactose is stereochemically different from glucose ■ Glucose is stabilized in enzyme by multiple H-bonds–can’t occur in galactose due  to different arrangement of -OH group ■ Induced fit change ■ Binding of substrate induces conformational change that forms active site ■ Example: binding of glucose to glucokinase ■ Lock and key fit involves less conformational change How do enzymes increase reaction rate?  ■ Reaction will reach same end point concentrations with or without enzyme ■ Reaction could take hours without enzyme to reach equilibrium, reaches  equilibrium in seconds with enzyme ■ Enzymes do not change Keq ■ Reactions plateau at equilibrium because… ■ Most/all substrate is used up ■ Enzyme is saturated with product ■ Forward reaction is occurring at same rate as reverse reaction ■ Activation energy=energy required to form transition state ■ Prevents molecule from falling apart ■ Activation energy does not factor into overall delta G ■ Enzymes lower the activation energy ■ Enzymes bind substrates, and stabilize them by noncovalent interactions■ Enzyme strains and distorts covalent bonds in substrate ■ Transition state occurs where bonds are most strained ■ In transition state, enzyme has maximum amount of noncovalent interactions with  substrate–stabilizes transition state and lowers activation energy ■ Enzyme also provides favorable microenvironment for catalysis ■ Active site is specifically designed to make noncovalent bonds with a specific  substrate’s transition state ■ 3 Phases of catalysis ■ 1) Binding of substrate E+S→ES ■ 2) conversion of substrate to product ES→EP ■ 3) Release of product EP→E+P ■ Enzymes return to original conformation and are not used up How can the reaction rate be measured?  ■ Rate of catalysis=moles of product formed/unit time ■ Determine initial velocity early in reaction–use to measure reaction rate ■ Michaelis-Menten equation–relationship between substrate concentration and velocity ■ k1=rate of formation of ES complex ■ k2=rate of dissociation of ES complex to E+S ■ k3=rate of conversion of ES complex to E+P ■ Michaelis-Menten assumptions: ■ ES complex rapidly forms and dissociates back to E+S ■ Breakdown of ES to E+P is the slowest rate ■ Michaelis Menten equation: Vi=Vmax[S]/Km+[S] ■ Km=(k2+k3)/k1 ←rate of ES dissociation v. rate of formation ■ Provides measure of ES complex strength ■ Vmax=k3[ET] ■ Maximum velocity at which the enzyme can catalyze a reaction ■ Depends on enzyme concentration ■ When [S]<Km, velocity is proportional to [S] ←addition of substrate will  increase reaction velocity linearly, first order kinetics ■ When [S]>>Km, velocity approaches Vmax, and substrate concentration does not  matter, zero order kinetics–enzyme is saturated ■ Km is a constant for a specific enzyme and a specific substrate ■ Estimate of dissociation constant of E from S ■ Enzyme will have more than 1 Km if it has more than 1 substrate ■ Concentration of substrate that gives ½ Vmax ■ Low Km=tight binding–rate of formation of ES is much faster than rate of dissociation ■ Low Km<10-9M ■ Advantages: enzyme can operate at low [S], substrate is still soluble, fewer  osmotic effects ■ High Km=weak binding ■ High Km~10-6M, need to increase [S] for activity, but has higher reaction rate ■ Example: Glucokinase v. Hexokinase ←both catalyze glucose-6- phosphate ■ Glucokinase has higher Km, used primarily in liver for storage, higher [S] ■ Hexokinase has much lower Km, used in other tissues for energy release,  lower [S] ■ Glucokinase responds to high levels of glucose after eating, when  hexokinase is already saturated ■ Liver helps regulate glucose levels ■ What if the brain has glucokinase? ■ Would not be able to process low glucose concentration between meals ■ Brain needs glucose constantly–problematic for high Km glucokinase ■ Alcohol metabolism example–Aldehyde dehydrogenase works more slowly in  people who can’t metabolize alcohol as well, causes worse symptoms ■ Kcat is turnover number for a given enzyme and substrate ■ Kcat=Vmax/[ET] ←not measure of catalytic efficiency ■ Represents how fast enzyme can catalyze at saturation ■ Units of moles product formed/sec at saturation ■ Catalytic efficiency=Kcat/Km ←Important because most enzymes are not at  saturation, takes this into account ■ Reveals preference of enzyme for substrate and stability of ES interaction ■ Depends on number and type of noncovalent interactions between enzyme and  substrate ■ Cancer drugs reduce catalytic efficiency of certain enzymes ■ Lineweaver-Burk Plot: 1/[S] vs. 1/[V0] ■ Only applies to Michaelis-Menten enzymes ■ Useful for determining inhibitor presence and mode of inhibition ■ Enzyme inhibitors–reversible vs. irreversible ■ Reversible: bind via noncovalent interactions ■ Competitive, noncompetitive, uncompetitive ■ Irreversible: bind covalently to active site, or via strong noncovalent interactions ■ Competitive inhibitors: Bind to active site reversibly, prevents access of substrate to active site ■ Increase Km (more substrate needed to reach ½ Vmax), but competitively  inhibited enzymes will still eventually reach Vmax at a very high [S] ■ Inhibitor will dissociate, substrate can bind if [S] is high ■ Holds true even if competitive inhibitor binds tighter than substrate ■ Competitive inhibitors resemble substrate ■ Examples of competitive inhibitors; ■ Methotrexate binds enzyme in place of tetrahydrofolate–enzyme  cannot form purines and pyrimidines–cancer cells can’t proliferate ■ Ritonavir–inhibits HIV protease ■ Chronic myelogenous leukemia treatment: block access of ATP to  tyrosine kinase ■ Noncompetitive inhibition–inhibitor binds to site on enzyme other than active site ■ Changes structure of active site, removes ability to form optimal  noncovalent interactions ■ Lowers Vmax, but Km remains unchanged ■ Dependant on concentration of inhibitor–if [I] is low, reaction can proceed ■ Example: Doxycycline–noncompetitive inhibitor of bacterial collagenase ■ Noncompetitive inhibitors can bind either to E alone or to ES complex ■ Uncompetitive inhibition–Similar to noncompetitive, but bind only to ES  complex, not E alone ■ Decreases Km and Vmax ■ Irreversible Inhibitors–involve covalent bond formation or strong noncovalent ■ Usually bind in active site with serine (-OH) or cysteine (-SH) ■ Decrease in enzyme activity over time ■ Some are suicide inhibitors–permanently deactivate enzyme, can only get more by  transcription ■ Example: Penicillin binds to serine in active site of transpeptidase ■ Is also transition state analog–binds more tightly to enzyme ■ Not all transition state analogs are irreversible, not all suicide inhibitors are  transition state analogs ■ Example: Aspirin–anti-inflammatory ■ Prostaglandin synthetase activity increases and causes increased  inflammation ■ Have long hydrophobic channel, need long fatty acid chain to make  prostaglandin ■ Fatty acid needs to enter channel for synthesis■ Aspirin binds to serine at entrance to channel, prevents access of fatty acid ■ NOT suicide or transition state inhibitor–can be broken down by body ■ Example: DIPF–organophosphate extensively used in pesticides ■ Binds acetylcholinesterase with serine -OH group in active site ■ Prevents serine from interacting with substrate ■ Results in too much acetylcholine in synapse–impairs muscle function and  breathing Allosteric Enzymes  ■ DO NOT FOLLOW MICHAELIS-MENTEN KINETICS!! ■ Relationship between [S] and V is sigmoidal rather than hyperbolic ■ Extremely important for regulating metabolism ■ Undergo conformational change, can bind non-substrate molecules to allosteric sites ■ Enzymes can have more than 1 allosteric site and more than 1 allosteric regulator ■ Binding of allosteric regulators influence R/T conformation ■ Have cooperativity and quaternary structure which contribute to sigmoidal kinetics ■ Example: PFK-1–uses ATP and fructose-6-phosphate as substrate to produce fructose 1,6  bisphosphate ■ Addition of fructose 2,6-bisphosphate (allosteric modifier) shifts curve to left,  makes curve more hyperbolic ■ Decreases amount of substrate needed to reach 50% max velocity ■ Example of positive allosteric modifier, allows PFK-1 to be active at lower [S] ■ Decreases K0.5, enzyme does not have to wait for substrate to build up ■ Shows that allosteric enzymes work on sliding scale of concentration depending  on regulators bound ■ Allosteric behavior is a form of enzyme regulation! ■ PFK-1 is major control point in glycolysis ■ Negative modifiers shift curve to the right, increase K0.5, makes curve more sigmoidal ■ Allosteric enzymes never fully inhibited, only slowed down, but never turned off ■ Example: ATCase ←+ modified by ATP, catalyzes chain that produces CTP,  which is a - modifier ■ Keeps balance between purine (ATP) and pyrimidine (CTP) in cell ■ Positive modifiers stabilize R state, increase enzyme activity ■ Negative modifiers stabilize T state, decrease enzyme activity ■ Negative modification of the beginning of a chain of reactions slows entire chain ■ Allosteric enzymes are sensitive to product accumulation ■ Concerted v. Sequential models for T→R transition ■ Concerted: all subunits are either T or R■ Substrate binding to 1 subunit changes conformation of all subunits ■ Disrupts T←→R equilibrium in favor of R–explains cooperativity ■ Explains sharp increase in enzyme activity ■ Sequential: Binding of substrate initiates conversion, but change happens  gradually ■ Allows for negative cooperativity–binding of substrate reduces affinity for  other substrates to bind Enzyme Regulation–Adaptation  ■ Hormones control metabolism, alter enzymatic activity ■ Enzyme will only produce as much product as the cell needs ■ Enzyme activity varies with physiological states ■ How are enzymes regulated? ■ Substrate concentration←Major mechanism ■ Enzyme concentration ■ Increase synthesis through gene activation–slow ■ Will not satisfy immediate needs of cell ■ Good for long term adaptation ■ Degradation–can happen very rapidly ■ However, if you need protein again, you need to start from scratch ■ Cyclins are most rapidly degraded proteins ■ Govern phases of cell cycle ■ Allosteric control–very rapid ■ +/- allosteric modifiers ■ Feedback inhibitors (CTP w/ATCase) ■ Reversible Covalent modification ■ Introduce phosphate to protein, affect electrostatic interactions due to -  charge ■ Change enzyme conformation and alter activity ■ Phosphate is added to hydroxyl group (Ser, Tyr, Thr) ■ Over 500 kinases catalyze these reactions, use ATP as substrate ■ Phosphatases reverse this reaction ■ Phosphorylation can either increase or decrease activity depending on  protein and pathway ■ Sequence surrounding Ser/Tyr/Thr recognized by different kinases,  allowing for specificity ■ Phosphorylation/dephosphorylation is initiated by external signal, like  hormone, cytokine, neurotransmitter, light, etc.)■ Signal is relayed to cell, eventually reaches kinase/phosphatase ■ Other covalent modification ■ Acetylation–add acetyl group to amino group of Lys or Arg–removes +  charge and decreases electrostatic interaction ■ Important in gene expression–causes histone proteins to relax hold  on DNA ■ ADP ribosylation–adds large, bulky, charged group–affects activity  significantly ■ Zymogen activators ■ Zymogen=larger precursor molecule, which is cleaved to form  active form of protein ■ Structure preserved after cleavage by disulfide bonds ■ Zymogen advantage–digestive enzymes cannot be active all the  time, otherwise they will digest stomach lining–only activated in  presence of food ■ Also seen in blood clotting proteins

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