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Chapter 10 Summery - Regulatory strategies

by: Kien Tran

Chapter 10 Summery - Regulatory strategies BCH 4024

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Kien Tran
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This note contains images and graphs an key points which I bet will help you have a complete understanding of this chapter
Dr. Orchieter
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This 10 page Class Notes was uploaded by Kien Tran on Sunday January 31, 2016. The Class Notes belongs to BCH 4024 at University of North Florida taught by Dr. Orchieter in Winter 2016. Since its upload, it has received 83 views. For similar materials see Biochemistry in Biology at University of North Florida.


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Date Created: 01/31/16
LEC15_EnzReg1 02/15/2007 05:12 PM Lecture 15: Enzymes: Regulation 1 [PDF] Reading: Berg, Tymoczko & Stryer, Chapter 10, pp. 275-282 Updated on: 2/15/07 at 5:00 pm Key Concepts Amounts of many key enzymes are regulated at the level of control of transcription, mRNA processing, and/or translation (mechanisms covered in BIOC 411 or BIOC 461), or destruction (proteolytic degradation) of old/unwanted enzymes. Activities of many key enzymes are regulated in cells, based on metabolic needs/conditions in vivo. Regulation of enzyme activity can increase or decrease substrate binding affinity and/or k . cat 5 principal ways protein activity (including enzyme activity) is regulated: allosteric control interaction with regulatory proteins multiple forms of enzymes (isozymes) reversible covalent modification irreversible covalent modification, including proteolytic activation Allosteric Control conformational changes -- 2 conformations, "R" (more active) and "T" (less active) allosteric activators (positive effectors/modulators) allosteric inhibitors (negative effectors/modulators) Allosterically regulated enzymes always multi-subunit ATCase as an example homotropic effector (substrate aspartate) heterotropic effectors (activator = ATP; inhibitor = CTP) 2 models for allosteric proteins MWC model (Monod-Wyman-Changeux, the "concerted" model): Either all subunits in a given protein molecule are in R state or all are in T state; no hybrid enzyme molecules. ATCase fits this model Koshland model (KNF model, the "sequential" model): includes possibility of "hybrid" enzyme molecules with some subunits in R state and others in T state. Hemoglobin (next lecture) requires elements of this more complicated model Objectives List 5 general strategies used by cells for biological regulation of enzyme activity. Explain the meaning of the “first committed step” in a metabolic pathway, and of the “rate-limiting step” in a metabolic pathway (which frequently turn out to be the same step), and explain the significance of those terms in regulation of “flow” of molecules through that pathway. Define/explain the following terms (some are review): quaternary structure, multimeric protein, homopolymeric protein, heteropolymeric protein, ligand, binding site, fractional saturation, feedback inhibition, cooperativity, cooperative binding, allosteric (allosteric site, allosteric effector/regulator, allosteric protein...), effector/regulator, homotropic effector/regulator, heterotropic effector/regulator, allosteric activator (positive heterotropic effector/regulator), allosteric inhibitor (negative heterotropic effector/regulator), protomer, prosthetic group. Briefly explain the allosteric regulation ofATCase, including its quaternary structure, its role in metabolism, and how its activity is regulated by allosteric inhibition and activation. Include the physiological rationale for the inhibition and activation. Sketch plots of V os. [S] for an allosteric enzyme that illustrate positive homotropic regulation and positive and negative heterotropic regulation, with ATCase as an example. Specifically, sketch (all on the same axes) for ATCase: V vs. [ospartate] curves with no heterotropic regulators present, with an allosteric inhibitor present, and with an allosteric activator present. Page 1 of 10 LEC15_EnzReg1 02/15/2007 05:12 PM Briefly explain the difference between the concerted model and the sequential model for an allosteric enzyme, in terms of R and T conformations of the individual subunits and of the whole quaternary structure, and whether “hybrid” quaternary structures (with some subunits in the T conformation and some subunits in the R conformation) exist in each model. 5 principal ways protein activity (including enzyme activity) is regulated: 1. Allosteric control Regulation of binding affinity for ligands, and/or of catalytic activity, by conformational changes caused by binding of the same or other ligands at other sites on protein ("allosteric effects") Changes involve simple association/dissociation of small molecules, so enzyme can cycle RAPIDLY between active and inactive (or more and less active) states. 2. Interaction with regulatory proteins Binding of a different protein to the enzyme alters the enzyme activity (activates or inhibits the enzyme) 3. Multiple forms of enzymes Isozymes (isoenzymes) = multiple forms of enzyme that catalyze same reaction but are products of different genes (so different amino acid sequences) Isozymes differ slightly in structure, and kinetic & regulatory properties are different Can be expressed in different tissues or organelles, at different stages of development, etc. 4. Reversible covalent modification Modification of catalytic or other properties of proteins by enzyme-catalyzed covalent attachment of a modifying group. Modifications removed by catalytic activity of a different enzyme, so enzyme can cycle between active and inactive (or more and less active) states. 5. Proteolytic activation Irreversible cleavage of peptide bonds to convert inactive protein/enzyme to active form. Inactive precursor protein = a zymogen (a proenzyme). Proteolytic activation IRREVERSIBLE, but eventually the activated protein is itself proteolyzed, or sometimes a tight-binding specific inhibitory protein inactivates it. 1. ALLOSTERIC REGULATION (Introductory material here is a review of concepts in chapter 7, ligand binding and allosteric regulation of hemoglobin, and won't be covered much in class.) Allosterically regulated enzymes: don't follow Michaelis-Menten kinetics (V vs. [S] is not a hyperbola.) o multisubunit enzymes (more than one catalytic subunit, so > 1 active site/enzyme molecule) cooperative substrate binding: binding of substrate to 1 active site affects properties of other binding sites (on other subunits) of the same enzyme molecule Result: a sigmoid Vovs. [S] curve (NOT hyperbolic) -- diagnostic of COOPERATIVITY Velocity increases steeply in [S] range around the apparent "KM" ([S] where velocity = 1/2 Vmax ), so in that substrate concentration range (around KM), a small change in [S] makes a big change in velocity. Regulatory consequences: binding another molecule (an allosteric inhibitor or activator) can SHIFT whole curve to RIGHT: Allosteric inhibitor --> velocity is less at a given [S], requires more [S] to reach 1/2max or SHIFT whole curve to LEFT: Allosteric activator --> velocity is greater at a given [S], requires less [S] to reach 1/max. Page 2 of 10 LEC15_EnzReg1 02/15/2007 05:12 PM Berg, Tymoczko & Stryer, 6th ed. Fig. 8.14: Kinetics for an allosteric enzyme, sigmoid V vo. [S] curve . MULTISUBUNIT proteins whose activity can be regulated by ligand binding-induced conformational changes Ligand binding to any protein is ligand concentration-dependent: Protein + n Ligand <==> Protein•Ligand n (ligand binding can be step-wise, 1 ligand molecule at a time) This reaction is described by an equilibrium constant at constant [P T concentration, ligand concentration determines position of equilibrium [L] acts as a signal that protein itself detects and responds to by a conformational change. Multisubunit protein has multiple binding sites for same ligand binding of "primary" ligand (substrate for an enzyme, O fo2 hemoglobin, etc.) can alter affinity of other binding sites on molecule for that same ligand (homotropic allosteric effects), and/or binding of other ligands (regulatory signaling molecules), to different sites from the primary ligand ("regulatory sites") can cause conformational changes that alter primary ligand binding affinity or catalytic activity (heterotropic allosteric effects). Sometimes regulatory sites are on different subunits (regulatory subunits) from binding sites for primary ligand. 2 examples of allosterically regulated proteins: hemoglobin (an O transport protein, not an enzyme) -- already covered in earlier lectures 2 aspartate transcarbamoylase (an enzyme) Regulatory enzymes in metabolic pathways usually fall into one or both of 2 categories: catalyze essentially irreversible metabolic reactions (LARGE NEGATIVE ΔG' under physiological conditions), AND/OR catalyze the FIRST COMMITTED STEP in a metabolic pathway Regulation of such enzymes permits efficient regulation of flux of metabolites through just THAT pathway. Page 3 of 10 LEC15_EnzReg1 02/15/2007 05:12 PM Examples of feedback inhibition: (left) Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 6-28: Feedback inhibition of threonine dehydratase (isoleucine biosynthetic pathway) (right) Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., Fig. 22-36 (abbreviated): Feedback inhibition of bacterial aspartate transcarbamoylase (ATCase) (pyrimidine nucleotide biosynthetic pathway) Aspartate transcarbamoylase ("ATCase"): catalyzes first committed step in the metabolic pathway for biosynthesis of pyrimidine nucleotides Nucleotides: compounds whose 3 covalently linked components are heterocyclic "base" (A, G, C or T in DNA; usually A, G, C or U in RNA) sugar (deoxyribose in DNA, ribose in RNA) phosphate building blocks of nucleic acids other major roles coenzymes energy storage compounds regulators of enzyme activity "Committed step": As a result of this step, metabolite (small molecule) is committed to continue down that pathway to endproduct No other branches lead to different endproducts that need to be regulated separately. FIRST committed step = the most efficient step for regulation of the rate -- that should also be the slowest step in pathway, controlling "flow" of matter to endproduct whose concentration you want to regulate. Regulation: Homotropic effects: aspartate (Asp) is bound cooperatively -- substrate binds preferentially to R state of ATCase. Page 4 of 10 LEC15_EnzReg1 02/15/2007 05:12 PM When Asp binds to active site on one subunit, Asp-binding affinity of active sites on other subunits in that same ATCase molecule increases. Result: sigmoid Vo vs. [Asp] plot Berg, Tymoczko & Stryer, 6th ed. Fig. 10.3: ATCase binds the substrate aspartate cooperatively (sigmoidal kinetics). Heterotropic effects: regulatory subunits of ATCase bind other ligands CTP (endproduct of whole pathway) is an allosteric INHIBITOR of ATCase. CTP binding to its sites on regulatory subunits → decreased binding affinity for Asp (substrate) to active sites on catalytic subunits. Lower affinity for Asp means apparent K for Asp increases, so at any given Asp concentration, V is decreased M o when [CTP] (endproduct concentration) is high. FEEDBACK INHIBITION :as endproduct of a metabolic pathway increases in concentration, cell needs to reduce rate of synthesis of that product Concentration of endproduct = a signal that "feed-back" inhibits the first committed step of pathway. example: CTP inhibition of pyrimidine biosynthesis Quaternary structure (subunit structure) of ATCase: Stryer, 4th ed., Fig. 10-4: Subunit arrangement in ATCase (catalytic subunits blue, regulatory subunits red) 6 catalytic chains total, arranged in 2 3 catalytic trimers 6 regulatory chains total, arranged in 3 r regulatory dimers 2 Catalytic trimers (c ) catalyze reaction in absence of 3 regulatory dimers, but for isolated catalytic trimers, Asp binding is NOT cooperative (so no communication between catalytic subunits in isolated trimer), and CTP has no effect on activity (not surprising -- CTP binds to the regulatory subunits). Berg, Tymoczko & Stryer, 6th ed. Fig. 10-6: Structure of ATCase Page 5 of 10 LEC15_EnzReg1 02/15/2007 05:12 PM Berg, Tymoczko and Stryer, 6th ed. Fig. 10.7: PALA, a bisubstrate analog resembles structure of a reaction intermediate binds tightly to active sites of ATCase Berg, Tymoczko and Stryer, 6th ed. Fig. 10.8: The 3 active sites in ATCase catalytic trimers are located at interfaces between pairs of c chains. Page 6 of 10 LEC15_EnzReg1 02/15/2007 05:12 PM position of tight-binding bisubstrate analog (PALA, phosphonacetyl-L-aspartate) resembles a reaction intermediate (shaded in gray) interacts also with some residues (boxed) from adjacent subunit, so active sites are at catalytic subunit interfaces Structures of free enzyme and enzyme with PALA bound determined by X-ray crystallography showed conformational change upon PALA binding. Compact, relatively inactive form: the T state ("tense") the expanded, more active conformation is called the R state ("relaxed"). Berg, Tymoczko and Stryer, 6th ed. Fig. 10.9: The T-to-R state conformational Berg, Tymoczko and Stryer, 6th ed. Fig. transition in ATCase. PALA binding (or normal substrate binding) stabilizes 10.11: The T-to-R state the R state, the more active form. conformational transition in ATCase. CTP binding stabilizes the T state, the less active form. Berg, Tymoczko and Stryer, 6th ed. Fig. 10.12: The T-to-R state conformational transition in ATCase. Even in the absence of any substrate or regulators, ATCase exists in an R-T equilibrium, with T state favored by a factor of about 200, i.e.{[T]/[R]} = about 200. eq Page 7 of 10 LEC15_EnzReg1 02/15/2007 05:12 PM Berg, Tymoczko and Stryer, 6th ed. Fig. 10.3: ATCase binds the substrate aspartate cooperatively (sigmoidal kinetics). Berg, Tymoczko and Stryer, 6th ed. Fig. 10.10 (below). The sigmoidal V os. [S] curve can be thought of as resulting from the mixture of the 2 conformational states, each following Michaelis-Menten kinetics (2 different hyperbolic o vs. [S] curves). T state predominates by a factor of about 200 at zero [S] has a very HIGH K M for Asp R state predominates at high [S]) has a much LOWER K M for Asp. As [Asp] increases so more Asp binds, enzyme shifts from T to R, so activity increases steeply, and apparent K M decreases, giving the sigmoidal plot. Page 8 of 10 LEC15_EnzReg1 02/15/2007 05:12 PM Berg, Tymoczko and Stryer, 6th ed. Fig. 10.13: CTP is an allosteric INHIBITOR, binding preferentially to the T state and thus stabilizing the T state (shifting the R-T equilibrium toward the T state), so V os. [S] curve shifts to right, as shown in red. Berg, Tymoczko and Stryer, 6th ed. Fig. 10.14: ATP is an allosteric ACTIVATOR of ATCase, preferentially binding to the R state, shifting the R-T equilibrium toward R state, which binds Asp more tightly, so curve shifts toward LEFT, as shown in blue. (For why ATP would "want" to be an activator, see below.) (Review concerted vs. sequential models for allostery from Chapter 7, allosteric regulation of hemoglobin.) concerted model = the Monod-Wyman-Changeux model = MWC or symmetry model: describes a protein or enzyme whose quaternary structure has only 2 forms, "all or none" Either all subunits in same molecule are in R state or all subunits in same molecule are in T state. Change in binding affinity for R <--> T conformational equilibrium can be modeled mathematically relatively simply, and fits observations for ATCase pretty well. You don't have to know the math below, just the concept. Berg, Tymoczko and Stryer, 6th ed. Fig. 10.15: Quantitative description of the MWC model for ATCase. Y = "fractional saturation", or "fractional activity" (enzyme) Y = [ES]/[E ] = [occupied sites]/[total sites] = V /V for an enzyme T o max α = ratio of [S] to Kdissocfor S from enzyme in R state (K ) R L = ratio [E ] / [E ] Tstate Rstate Binding of allosteric effectors (CTP, inhibitor; and ATP, activator) to ATCase changes L, and thus changes response to [S]. Page 9 of 10 LEC15_EnzReg1 02/15/2007 05:12 PM Data for ATCase fit MWC model for which in absence of heterotropic effectors, T state / R state ratio = about 200 / 1. Inhibitor (CTP) increases T/R ratio (so fewer enzyme active sites in more active R conformation). Activator (ATP) decreases T/R ratio (so more active sites in the more active R conformation). Why is it metabolically useful for ATP to be an allosteric ACTIVATOR for ATCase, OVERRIDING CTP feedback inhibition of ATCase? CTP (end product of the pyrimidine nucleotide biosynthetic pathway) = a feedback inhibitor reduces the rate of flux through pathway and thus slows formation of more pyrimidine nucleotides when CTP concentration is already high ATP (a purine nucleotide) activates ATCase and thus leads to more pyrimidine biosynthesis WHY? Purine nucleotides are also needed for nucleic acid biosynthesis. ATP is a compound used to "store" metabolic energy in the cell. High concentration of ATP is an intracellular indicator that the cell is energy-rich, very "happy" metabolically. High [ATP] concentration thus "tells" the cell a) there are lots of purine nucleotides available, so you should go on making pyrimidine nucleotides to keep nucleotide pool balanced so we don't run short of either kind for nucleic acid biosynthesis, and b) cell is in great shape metabolically and wants to replicate its DNA and divide, so high concentration of nucleotides is needed. High [ATP] thus can "override" inhibitory signal of high [CTP] and activate ATCase. NEXT LECTURE: Other modes of enzyme regulation: isozymes, covalent modification, regulatory proteins, proteolytic cleavage. Department of Biochemistry & Molecular Biophysics The University of Arizona Copyright (©) 2007 All rights reserved. Page 10 of 10


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