StudyGuide2 Biochemistry I
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This 16 page Study Guide was uploaded by Spurthi Pasham on Saturday July 16, 2016. The Study Guide belongs to Biochemistry I at University of Texas at Dallas taught by Mehmet CandasJiyong Lee in Summer 2016. Since its upload, it has received 37 views. For similar materials see Biochemistry I in Biochemistry at University of Texas at Dallas.
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Date Created: 07/16/16
TOPICS • Protein Secondary, Tertiary and Quaternary Structure; Chapter 6 • Enzyme Kinetics; Inhibition Kinetics; Irreversible Inhibition; Bisubstrate Kinetics; Chapter 13 • Enzyme Mechanisms; Chapter 14 Please update study guide as per review session .1. What is the configuration of peptide bonds in proteins? “Alpha carbons of adjacent residues are almost always in trans configuration” Ch. 6, slide 14 Otherwise there is steric hindrance. Trans is lower energy configuration. The configuration of peptide bonds in proteins refers to the 1' amino acid sequence .2. How is the conformation of the backbone of a polypeptide described? “The angle about the a CN bond is denoted by Greek letter phi , and angle about the aCoC is denoted by psi… entire path of peptide backbone is protein is known if the phi and psi rotation angles are specified. Some values of phi and psi are not allowed due to steric interference between nonbonded atoms” Ch. 6, pg. 137 .3. What does Ramachandran plot display? aka [φ,ψ] plot Shows sterically reasonable values of the angles phi and psi. “Most combinations of phi and psi are sterically forbidden, and the corresponding regions of the Ramachandran plot are sparsely populated” Ch. 6, pg. 13 7 4. What is rise and pitch on helix axis of a polypeptide? How do they affect the helical turn number on a polypeptide with a given number of amino acids? Rise= how far the helix is going up, esp. per residue, (advances per AA residue) (1.5 Angstroms or 0.15 nm) Pitch= rise/turn (advances per turn) (0.54 nm or 5.4 Angstroms) “The backbone loop closed by any Hbond in an alpha helix contains 13 atoms”chapter 6 lecture slides. So small rise values should take up less physical space, but the turn numbers would be the same for a given number of amino acids. When more helical strands intertwine, the number of residues per turn decreases for example, collagen (three intertwined alpha helixes) only has 3.3 residues per turn, so it’s much more stretched out than an alpha helix by itself. .5. Why proline is not often found in αhelices of proteins? Proline and glycine are commonly classified as “helixbreakers”, but proline is the only amino acid that only operates as a helix breaker. (To whomever was curious to why glycine is also considered a helix breaker, it is because it is so flexible that it ends up disrupting the helix) Proline has a five member nitrogen containing ring. Its side chain connects to protein backbone twice, and it can’t occupy main chain conformations as easily as other amino acids can. It can introduce kinks into alpha helices because it can’t adopt normal helical conformations. .6. What is leucine zipper? What type of proteins exhibit leucine zipper structure? The leucine zipper is a common threedimensional structural motif in proteins and it has that name because leucines occur every 7 amino acids in the dimerization domain. The localization of the leucines are critical for the DNA binding to the proteins. Leucine zippers are present in both eukaryotic and prokaryotic regulatory proteins, but are mainly in eukaryotes. Leucine Zippers are a class of proteins that bind to DNA at specific sites within the promoters of genes. When the protein is bound to the promoter, transcription is stimulated and the gene is "expressed". This class of DNA binding proteins gets its name from the regular pattern of leucine residues within the two alpha helices. Being hydrophobic, the leucines cause two adjacent alpha helices to be "zippered" together by hydrophobic interactions. .7. What are the principal forces holding subunits of an oligomeric protein? Hydrophobic interactions .8. How can we experimentally estimate the molecular weight of oligomeric proteins? What is the methodology? Gel filtration chromatography when an aqueous solution is used to transport the sample through the column; results in the fractionation of proteins and other watersoluble polymers. This is separation based on size. → is less sensitive way to determine the molecular weight of the assembly .9. How can we experimentally determine the molecular weight of each chain of oligomeric proteins? SDS determines weight of each chain. Sds gel electrophoresis ch.5 → To separate polypeptide chains and estimate molecular weight, is considered more accurate .10. What is the methodology? .11. Why do many proteins have multiple subunits? 1. Because they create overall a more stable structure 2. Genetic economy and efficiency 3. Cooperativity 4. Bring catalytic sites together 12. What are proteins segments? What is their role in protein folding? The segments of a protein that are not helices or sheets are traditionally referred to as “random coil”, although this term is misleading: •Most of these segments are neither coiled or random •They are usually organized and stable, but don’t conform to any frequently recurring pattern •Random coil segments are strongly influenced by sidechain interactions with the rest of the protein The conformational entropy associated with the randomcoil state significantly contributes to its energetic stabilization and accounts for much of the energy barrier to protein folding. Protein segments are small bits of proteins that frequently have the same amino acid sequence that affect how the protein folds. For instance, a segment of hydrophobic amino acids is going to fold inwards due to hydrophobic forces, which alters the shape of the entire protein. 13. What is the function of chaperone proteins? Molecular Chaperones Are Proteins That Help Other Proteins to Fold. They protect nascent proteins from the concentrated protein matrix in the cell and perhaps to accelerate slow steps. Chaperone proteins were first identified as "heatshock proteins" (Hsp60 and Hsp70) .14. How do proteins fold fast? Funnellike energy landscape example: at the top=unfolded proteins, once polypeptide start falling down the funnel walls results in the 1st contact between the residues that establish different folding possibilities. Then partial intermediates are formed representing the tertiary structure. Once the polyp. Came to the bottom, it forms the folding structure which is the quaternary structure. Proteins fold rapidly into correct, minimal energy configurations because amino acids interact locally due to physiological and chemical properties Cooperativity limits conformation spaces protein has to explore (otherwise it would take way too long) and forces them to follow funnel like energy landscape that allows it fold quickly. 15. How do reactions progress in terms of free energy? When gibbs free energy is negative ( spontaneous), the folded state is more stable than the unfolded. Therefore, the folded state is preferred. As gibbs free energy decreases it allows the proteins to fold more. 16. What do the peaks and valleys of an energy vs reaction progression plot correspond? Represents the graph free energy vs progress of reaction from ch 13. And how the free energy of activation for the reaction it is greater without an enzyme involved. Peaks = transition states Valleys = Intermediates 17. How are the favorable and unfavorable reactions defined in terms of free energy? Favorable =when delta G is negative (exergonic) Unfavorable = when delta G is positive (endergonic) .18. What is meant by diffusioncontrolled reaction? Aka Diffusion limited enzyme catalysis •A diffusion limited enzyme catalyzes a reaction so efficiently that the rate is limited to substrate diffusion into the active site, or product diffusion out. •This is also known as kinetic perfection or catalytic perfection. •Diffusioncontrolled reaction, therefore represents an intrinsic, physical constraint on evolution. In other words, there is an apparent maximum peak height in the fitness landscape. No other factors really affect the diffusion other than the substrate/ligand concentration in the active site; most efficient possible .19. What is reaction order? Reaction order is the index or exponent to which the concentration term of the rate equation is raised to Hanes woolf equation .20. How is the reaction velocity described according to reaction order? Zeroth Constant First Linear Second Exponential .21. How do enzymes accelerate reactions? “Enzymes lower ΔG‡ [free energy of activation], thereby accelerating rate.” (Ch 13, slide 6) They stabilize the transition state, thus lowering the activation energy. .22. What is the lock and key model of enzyme catalyzed reactions? The “Lock and key” hypothesis was the first explanation for specificity. Only the correctly sized key (substrate) fits into the keyhole (active site) of the lock (enzyme). The binding of the substrate to the enzyme causes a change in conformation. .23. Why does the formation of an enzymesubstrate complex tend to be thermodynamically favorable? Desolvation is the process where in an aqueous solution containing an enzyme and a substrate, water that is surrounding the substrate is replaced by the enzyme. In other words, water molecules that were once in between the substrate and the enzyme are displaced to allow the interaction of the substrate with the enzyme. The process also increases the entropy of the reaction, making the formation of the enzymesubstrate complex more thermodynamically favorable. (I thought formation of ES resulted in a loss of entropy, causing destabilization of ES and therefore increasing the rate of reaction and making it thermodynamically favorable? Ch 14) •Enzymes catalyze thermodynamically favorable reactions, causing them to proceed at extraordinarily rapid rates •Enzymes provide cells with the ability to exert kinetic control over thermodynamic potentiality .24. What is the MichaelisMenton equation? .25. What characteristics of enzyme catalysis can be learned from MichaelisMenton plot? Km and Vmax .26. What are the linear transformations of the MichaelisMenten curve? Take the reciprocal of the MichaelisMenton equation and you get a LineweaverBurk plot. Or multiply lineweaver burk by [S] and get a HanesWoolf plot. .27. Why is the linear transformation of the MichaelisMenten curve useful? Linear transformations are easier to read, draw, and use. .28. What does KM describe for an enzyme catalyzed reaction? •The "kinetic activator constant" •Km is a constant derived from rate constants •Km is, under true MichaelisMenten conditions, an estimate of the dissociation constant of E from S •Small Km means tight binding; high Km means weak binding KM is the MM constant; it is the concentration at which the rate of the process equals half of the maximum rate. .29. What are the mechanisms of enzyme catalysis? •Enzymes facilitate formation of nearattack complexes which is the precursor for the transition state •Protein motions are essential to enzyme catalysis •Covalent catalysis phosphorylation •General acidbase catalysis •Lowbarrier hydrogen bonds LBHBs can occur when the pKa of the two heteroatoms are closely matched, which allows the hydrogen to be more equally shared between them. the formation of a LBHB could form during catalysis to stabilize a transition state •Metal ion catalysis specific mechanism that utilizes metalloenzymes with tightly bound metal ions such as Fe , Cu , Zn , Mn , Co , Ni , Mo (the first three being the most commonly used) to carry out a catalytic reaction .30. What groups on amino acids can make good nucleophilic catalyst? The side chains. These groups readily attack electrophilic centers of substrates, forming covalent enzymesubstrate complexes. Includes amines, carboxylates, aryl and alkyl hydroxyls, imidazoles, and thiol groups Serine, Cysteine, and Threonine 31. What is acidbase catalysis in enzyme reaction? Catalysis in which a proton is transferred in the transition state. "Specific" acidbase catalysis involves H+ or OH that diffuses into the catalytic center. "General" acidbase catalysis involves acids and bases other than H+ and OH. These other acids and bases facilitate transfer of H+ in the transition state. .32. Why can the imidazole side chain of histidine can function as either a general acid catalyst or a general base catalyst? Because the pKa is so close to neutral (amphoteric). At physiological pH range, the nitrogen ring can be easily protonated/deprotonated .33. What is the reaction equilibrium in enzyme catalysis? The rate of catalysis rises linearly with substrate concentration but levels off at higher concentrations. Saturation effect→ V doesn’t increase even when substrate concentration increases because the active site is saturated and every enzyme molecule has its active site occupied with substrate. k1=k2 34. How will the pH vs. velocity curve look if an enzymecatalyzed reaction requires a group with a low pK and a group with a higher pK? Enzymatic Activity is Strongly Influenced by pH •Enzymesubstrate recognition and catalysis are greatly dependent on pH •Enzymes are usually active only over a limited range of pH •The effects of pH may be due to effects on Km or Vmax or both The optimal pH is different for every reactant. The curve would reach a maximum at a pH within it’s ideal functioning range and decrease at higher pHs due to denaturation? 35. What type of amino acids are involved in the catalytic step of an enzyme reaction if pH vs. velocity curve displays an inflection point at pH~4? Acidic; A pH vs. Velocity curve with inflection point at pH 4 will have an amino acid on the active site with a pka of 4 because inflection points on the curve occur at the pka’s of the amino acid. Inflection points on pH vs. velocity curves occur at the pKas of the amino acids, so a pH vs. velocity curve with an inflection point at pH 4 will have an amino acid on the active site with a pKa of 4 36. What are the physical determinants of reaction rate in terms of reacting groups involved in a given enzymatic catalysis? Concentration of reacting molecules with transition state energy (slide 25, ch 13) 37. What are the differences between competitive, uncompetitive, noncompetitive, and irreversible enzyme inhibition? How do different inhibition types change the 1/V vs 1/[S] plot? Ch 13 slides 6164 Competitive Inhibitors Compete With Substrate for the Same Site on the Enzyme same yintercept as uninhibited enzyme, since Vmax is not affected by competitive inhibition, 1/vmax does not change different slopes and xintercepts because the inhibitor is similar to substrate, the substrate can’t be acted on by enzyme and there are fewer active sites available for it to bind to need to increase substrate concentration in order to achieve reaction rate Pure Noncompetitive Inhibition – where S and I bind to different sites on the enzyme. Note that I does not alter Km but that it decreases Vmax. Mixed Noncompetitive Inhibition: binding of I by E influences binding of S by E. Note that both intercepts and the slope change in the presence of I. can’t overcome by increasing substrate concentration enzyme substrate inhibitor complex (new entity form) doesn’t go to products form decreases efficiency of reaction, changes processivity, not Mm concentration (?) Uncompetitive Inhibition, where I combines only with ES, but not with E. Note that both intercepts change but the slope (Km/Vmax) remains constant in the presence of I. Enzymes Can Be Inhibited Irreversibly. Penicillin is an irreversible inhibitor of the enzyme glycoprotein peptidease, which catalyzes an essential step in bacterial cell wall synthesis. form “permanent” covalent bonds with side chains or prosthetic groups in enzyme decreases concentration of active enzyme can be distinguished from noncompetitive b/c reaction of I with E is not instantaneous time dependency decreases enzymatic activity `dilution of enzyme: inhibitor solution doesn’t dissociate EI complex/restore enzyme activity .38. What are transitionstate analogs? What is their affinity towards their interaction with reaction components? Transition state analogs (TSAs) are stable molecules that are chemically and structurally similar to the transition state Purine riboside inhibits adenosine deaminase. The hydrated form is an analog of the transition state of the reaction. TSA’s have a higher affinity for enzymes because enzymes have an especially favorable interaction with transition states in a particular reaction. Since the TSA mimics the transition state, enzymes have a higher affinity for the TSA. .39. What is a competitive inhibitor? How does it affect Vmax of an enzyme catalyzed reaction? Inhibitor molecule similar to the substrate but unable to be acted on by the enzyme competes with the substrate for the active site. Because of the presence of the inhibitor, fewer active sites are available to act on the substrate. Higher substrate concentration is required to achieve a given reaction rate. Competitive inhibitors have the same yintercept as uninhibited enzyme (since Vmax is unaffected by competitive inhibitors the inverse of Vmax also doesn't change) but there are different slopes and xintercepts between the two data sets. Compete with the substrate for the same active site on the enzyme 40. What is an irreversible enzyme inhibitor? How does it bind to the enzyme? Irreversible inhibitors bind permanently to their target enzyme, often via a covalent bond that influences catalysis. "Permanently" here means over a timescale that is long compared to the functional lifetime of the enzyme itself; that timescale may be minutes for some bacterial enzymes, and months or years for enzymes found in stationary populations of cells in eukaryotes. Irreversible inhibitors can give us some understanding of enzyme function, and they can be employed as pharmaceuticals, but they are less informative and less common than reversible inhibitors. Penicillin is an irreversible inhibitor of the enzyme glycoprotein peptidease, which catalyzes an essential step in bacterial cell wall synthesis. It binds to the peptide bond, but the enzyme remains inactive. 41. What is the uniqueness of enzyme catalyzed reactions in comparison to typical chemical catalyzed reactions in organic chemistry? Enzymes are highly specific and produce large amounts of product while chemical catalysts are less specific and can produce more errors -biological specificity, occur batch wise, and have 2-3 orders http://www.diffen.com/difference/Catalyst_vs_Enzyme .42. What is the MichaelisMenton constant? KM is the MM constant; it is the concentration at which the rate of the process equals half of the maximum rate. .43. Which step in an enzymecatalyzed reaction was assumed to be negligible by Michaelis and Menton? Formation of ES from E + P (backward) k2 .44. What are the assumptions made in calculating the MichaelisMenten Equation? assumes the formation of an enzymesubstrate complex assumes that the ES complex is in rapid equilibrium with free enzyme assumes that the breakdown of ES to form products is slower than 1) formation of ES and (k1>k2) 2) breakdown of ES to reform E and S” (Ch 13, slide 33) ( k2 < k1) 45. Can you calculate the value of the maximum velocity for an enzymecatalyzed reaction that follows MichaelisMenton kinetics if the initial velocity, substrate concentration, and KM for the enzyme system is given? Vmax= Vo((Km+[S])/[S]) 46. What are the differences in terms of substrate binding and processing between random, sequential, ordered, and pingpong reaction mechanisms? Sequential, or Singledisplacement reactions can be of two distinct classes: 1. Ordered, where a leading substrate binds first, followed by the other substrate 2. Random, where either substrate may bind first, followed by the other substrate DoubleDisplacement (PingPong) reactions proceed via formation of a covalently modified enzyme intermediate. Product of enzyme reaction with A is released before enzyme reacts with second substrate; there are two half reactions and the substrates don’t react directly with each other 47. Do allosteric enzymes follow typical MichaelisMenten kinetics? No, because they don’t bind to the active site, rather they bind to the allosteric site. .48. What happens to the rate of an enzymecatalyzed reaction if substrate depleted steadily with time? The rate on an enzyme catalyzed reaction is proportional to the substrate. The rate decreases linearly as substrate concentration decreases and then begins to level off and approach a minimum. If the substrate concentration of an enzymecatalyzed reaction decreases steadily with time, the reaction rate drops very rapidly (think quadratic or cubic rather than linear) 49. How is the rate of breakdown of the enzymesubstrate complex denoted? The rates of formation and breakdown of the ES complex are given in terms of known quantities: The rate of formation of ES =k1[E][S] (with the assumption that [P] =0) The rate of breakdown of ES = k 2[ES]+k 3[ES]=(K 2+K 3)[ES] The rate of breakdown of the enzyme substrate complex is denoted as k2 (the formation of it is k1) .50. What the steady state assumption is as applied to enzyme kinetics? the ES complex is formed and broken down at equivalent rates. (forward rate = reverse) k1=k2 51. What type of amino acids can be found on the surface of a singlesubunit protein? Give examples. Hydrophilic amino acids Polar AA: Glutamine, Asparagine, Serine, Threonine, Cysteine, Methionine 52. Which amino acid is the most conformationallyrestricted? Proline 53. What are the corresponding energy levels of reactant, transition state, intermediate, and product on an energy diagram? Ch 13 (Really general question) 54. Which amino acid(s) is/are least likely to participate in acidbase catalysis? , it should be any nonpolar, hydrophobic amino acids such as alanine(most unreactive) or leucine. Amino acids that don’t have acidic or basic side chains: the amino acids with aliphatic and aromatic side chains and the amino acids with polar neutral side groups 55. Why is histidine an ideal amino acid at neutral pH values at the active site of many enzymes? An activesite histidine, which might normally be protonated, can be deprotonated by another group and then act as a base, accepting a proton from the substrate. 56. What is the use of methodology called sitedirected mutagenesis? Sitedirected mutagenesis (SDM) is a method to create specific, targeted changes in double stranded plasmid DNA. There are many reasons to make specific DNA alterations (insertions, deletions and substitutions), including: To study changes in protein activity that occur as a result of the DNA manipulation. To select or screen for mutations (at the DNA, RNA or protein level) that have a desired property To introduce or remove restriction endonuclease sites or tags 57. If an enzyme's active site contains only two ionizable residues, an arginine and a glutamate (pKa's of 2.9 and 9.1, respectively) that participate in the catalytic mechanism, what would you expect for the optimum pH of the enzyme? A pH midway between 2.9 and 9.1 (pH = 6), where arginine is deprotonated and glutamate is protonated. (answer confirmed in lecture)
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