Class Note for BIOC 460 at UA
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Date Created: 02/06/15
BIOC 460 Spring 2008 Lectu re 1 5 Enzymes Regulation 1 Allosteric Regulation lsozymes Subsequent 2 lectures Reversible covalent modification Association with regulatory proteins Irreversible covalent modificationproteolytic cleavage Reading Berg Tymoczko amp Stryer 6th ed Chapter 10 pp 275283 Key Concepts Amounts ofmany key enzymes are regulated at the level of control of transcription mRNA processing andortranslation mechanisms covered in BIOC 411 or BIOC 461 or destruction proteolytic degradation of oldunwanted enzymes Activities ofmany key enzymes are regulated in cells based on metabolic needsconditions in vivo Regulation of enzyme activity can increase or decrease substrate binding affinity andor km 5 ways to regulate protein activity including enzyme activity allosteric control multiple forms of enzymes isozymes reversible covalent modi cation interaction with regulatory proteins irreversible covalent modi cation including proteolytic activation J F P NT Catalyze essentially irreversible metabolic reactions AG39 large neg andor Catalyze the first committedstep in a metabolic pathway Regulation of such steps permits ef cient regulation of flux of metabolites through just that pathway quotCommitted stepquot As a result ofthis 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 ef cient step for regulation of the rate that should also be the slowest step in pathway controlling quot owquot of matter to endproduct whose concentration you want to regulate Feedback inhibition endproduct acts as an allosteric inhibitor of enzyme camlyzing FIRST COMIVJIITED STEP in that pathway 3 E a F3 em pm uct E E 1 E1 3 A AB 4 C 4 D 15gt Q 4 R 4 s endproduct 8 E7 Es Key Concepts continued T Learn39ns ObJeCt39Ves erminology some are reVIew quaternary structure multimenc protein 1 Allosmric mm homopolymeric protein heteropolymeric protein ligand binding site conformational Changes fractional saturation feedback inhibition cooperativity cooperative 2 conformations in equilibrium quotRquot more active amp quotTquot less active binding allosteric homotropic effectorregulator heterotropic allosteric activators positive effectorsmodulators effectorregulator allosteric activator positive heterotropic allosteric inhibitors negative effectorsmodulators e eCtOFregUator EIIOSteriC iNhibitor quoteg tiVe heterOt39OPiC o en feedback inhibitors product of pathway inhibits first effectorregulator protomen prosthetic group isozyme committed step in pathway Brie y explain the allosteric regulation ofATCase including its quaternary Anestericauy reguiated enzymes aiways muitpsubunit structure its role in metabolism and how its activity is regulated by Aspartame transcarbamoylase InCase as an example allosteric inhibition and activation Include the physiological rationale for the inhibition and activation homotropic effector activator substrate aspartate heterotropic effectors activator ATP inhibitor CTP 39 kemh pk Ofv Ys39 s for an aHOStenc enzyme that quotlusz posmye omotropic regulation and positive and negative heterotropic regulation 239 l5 zymes with ATCase as an example Speci cally sketch all on the same axes Multiple forms of an enzyme that catalyze the same reaction for ATCase Vo vs aspartate curves with no heterotropic regulators Different kinetic parameters ke Km andor different anosten39c present with an allosteric inhibitor present and with an allosteric activator regulation with physiological consequences Present Hexokinase different forms in livervs muscle re ect the different 39 Explain the bi 09i al usemmess fi5 Zym95i arid discuss the example 0f roes ofthose tissues in the body muscle hexokinase vs liver glucokinase in terms of difference in Jnction ofthe tissues I I Regulatory enzymes 5 principal ways proteinenzyme activity is regulated n genera Allosteric control Regulation of binding af nity for ligands andor of catalytic activity by conformational changes caused by binding of the same or other ligands at othersites on protein quotallosteric effectsquot Changes involve simple associationdissociation 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 usually by causing conformational change 3 Multiple forms of enzymes isozymes lsozymes isoenzymes multiple forms of enzyme that catalyze same reaction but are products of different genes so different amino acid sequences lsozymes differ slightly in structure and kinetic and regulatory properties are different Can be expressed in different tissues or organelles at different stages of development etc LEC 15 Enzymes Regulation 1 BIOC 460 Spring 2008 5 principal ways proteinenzyme activity is regulated 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 proteinenzyme to active form Inactive precursor protein a zymogen a proenzyme Proteolytic activation irreversible but eventually the activated protein is itself proteolyzed or sometimes a tightbinding specific inhibitory protein inactivates it Allosteric Regulation Multisubunit enzymes more than one active site per enzyme Regulation of binding affinity for ligands like substrates andor catalytic activity km Conformational changes linked with ligand binding homotropic effects binding of quotprimaryquot ligand substrate for an enzyme 02 for hemoglobin etc can alter affinity of other binding sites on molecule for that same ligand heterotropic effects binding of other ligands regulatory signaling molecules to different sites from the primary ligand quotregulatory sitesquot can cause conformational changes that alter primary ligand binding affinity or catalytic activity Sometimes regulatory sites are on different subunits regulatory subunitsquot from binding sites for primary ligand Ligand bindinginduced conformational changes Ligand concentration signal cell needs more or less of some metabolic product Signal detected by regulated enzyme Allosteric regulation permits rapid cycling of enzyme between more active and less active conformations just associationdissociation of small molecules Allosteric activators gt higher activity Homotropic effectormodulator substrate itself Heterotropic effectors eg Metabolite earlier than substrate in same pathway feedahead activation Other metabolites ligands that act as indicators of metabolic need Allosteric inhibitors gt lower activity Heterotropic effectorsmodulators Product of whole pathway feedback inhibitionquot another ligand that acts as indicatorthat cell needs less of that pathway s product Homoallostery Enzymes that are allosterically regulated do NOT follow Michaelis Menten kinetics Vo vs 5 curve is not hyperbolic cooperative substrate bindingactivation cooperativity 8 binding to one active site alters 8 binding affinity andor catalytic activity at other active sites on same enzyme molecule Sigmoid Vo vs 5 curve Nanceoperative Enzyme 050 Allosteric Enzyme S Heteroallostery Regulation by heterotropic effectors can be positive activation favors R state or negative inhibition favors T state Heterotropic effectors bind to different site from active site 100 Positive effector 075 gt Basal rate 050 Negative effector 025 4 Physiological S 000 39 39 39 0 25 50 75 100 S Aspa rtate transca rbamoylase Pyrimidine nucleotide biosyuthesis quotAreasev catalyzes first committed step in the metabolic pathway for Inl m slzgtilhessis of pyrimidine j ATCRSE Nucleotides Pi compounds whose 3 covalently Ncarbamoylaspartate linked components are heterocyclic quotbasequot A G C l or T in DNA usually A G c I 5 feedback or U In RNA iinhibition sugar deoxyribose in DNA 5 ribose in RNA 5 phosphate l building blocks of nucleic acids UMP i other major roles coenzymes energy storage compounds UTP regulators of enzyme activity 4 E CTP 7777777777 77 LEC 15 Enzymes Regulation 1 BIOC 460 Spring 2008 ATCase reaction Condensation of Asp carbamoyl phosphate gt carbamoyl aspartate P 0 o 0 NH 1 NH CH l H 2 0 k C P o ovog HN 0 zAcoo orbnmnyl Aspnvlole Nnllmmoylusponme phosphate Hz NJ 0 o 2 r 0 gt N 0 0 o o 0 0 endproduct ofpathwgy CTP HO OH Berg 9 5 F g 10quot ylidine Iriplmspllnla a PALA a bisubstrate analog for ATCase Binds to active site of R state active conformation of ATCase Can t react to form products 0 c39o o HzC H chr N 2 I 1 a CoPoJ h F03 H2 NH2 quotODC NH 1 H7N 39OOC Reunion intermediate Note resemblance of PALA to reaction intermediate 9 PO 139 A CE H NlPIIospholImeOyllluspu ute PAM Bound substrates Berg etai Frg 1077 Homoallostery in ATCase In absence of any substrate or regulators ATCase RT equilibrium favors T state by a factor of about 200 To R0 20 ATCase binds the substrate aspartate cooperatively sigmoidal kinetics T state predominates by a factor of about 200 at zero 8 has a very HIGH Km for Asp R state predominates at high 8 has a much LOWER Kquot for Asp Rate of Narbamoylaspartale formation gt 10 20 30 40 AspartateL mM ATCase Substrate Binding cooperative substrate binding mixture of R and T states Equilibrium at very low 8 lies far toward T conformation At zero 8 T stateR state 2001 for ATCase Equilibrium shifts from T toward R as more 8 binds T state less active usually lower binding af nity for 8 higher KEl 5 R state more active usually higher af nity for 8 lower KB 5 ATCase activity Rstate curve When Asp binds to active site on one subunit Asp binding af nity of active sites on other subunits in that same ATCase molecule increases As Asp increases so more Asp binds enzyme shifts from T to R so activity increases steeply and apparent Km decreases giving the sigmoid VEl vs Asp plot Trstate curve Rate of Ncarbamoylaspartate formation gt Berg et ai Fig i040 Aspartate gt Structural basis for allosteric regulation in ATCase Quaternary structure subunit structure ofATCase 12 subunits 6 catalytic chains total arranged in 2 c3 catalytic trimers blue 6 regulatory chains total arranged in 3 r2 regulatory dimers red Regulatory subunits bind heterotropic effectors CTP feedback inhibitor and ATP allosteric activator Catalytic trimers ca catalyze reaction in absence of regulatory dimers For isolated catalytic trimers ca 3 substrate Asp binding is NOT cooperative no communication between catalytic subunits in ca Feedback inhibitor CTP has no effect on activity of isolated catalytic trimers not surprising CTP binds to regulatory subunits stryer 4th ed Frg 104 Another view of ATCase structure Zin Regulatory M rmain c hain Calalylic Bl trimer Regulatory dimer Regylatory Side View dlmer Regulatory dimer Catalytic llimer Berg etai Frg 1076 LEC 15 Enzymes Regulation 1 BIOC 460 Spring 2008 ATCase active sites at interfaces between catalytic subunits Quaternary structural changes T gt R in ATCase 39 3 acme f39tes per cataly c mrner T state tense form more R state relaxed form PAIA bisubstrate analog binds very tightly Interacting With compact less active expanded more active residues on both sides of subunit interfaces favored by PALA binding PALA a bisubstrate analog mm 6A and by ATP binding subunit WT Avg 161 1 His 134 5 PALA Gin 231 a g 3 Avg w r a i u a a 7 I 0 5 4 a w miss 339 vs a 24 quot Mg 219 f F x j as 3 y 5 a E Th753 5 A 1 slate I state I Berg etal Fig 109 gt 39 T state less active Quaternary structural changes T R in ATCase stabilized by CTP binding In absence of any substrate or regulators ATCase RT equilibrium favors T state by a factor of about 200 CTP is a feedback inhibitor T I R 200 the endprodu ct of the pathway of pyrimidine nucleotide 1 5 R 5quotquot biosynthSSIS T 5quotquot less active more active CTP E i Favored by CTP binding Favored by substrate binding Berg em F g 1039 T 5quotquot Berg etal Fig 1012 Heterotropic Effects in ATCase Heterotropic Effects in ATCase Heterotropic ligands bind to the regulatory subunits of CTP is an allosten39c inhibitor ofATCase ATCase binds preferentially to T state thus stabilizing T state CTP endproduct of whole pathway allosteric inhibitor of shi s RT equilibrium tOWard T state so VB vs 8 curve shi s to right ATCase as shown in red binds preferentially to T state ofwhole ATCase thus decreases binding af nity for Asp substrate to active sites on catalytic subunits Lower af nity for Asp means apparent Km for Asp increases so at any given Asp concentration VB is decreased when CTP is high FEEDBACK INHIBITION As endproduct of a metabolic pathway increases in concentration cell needs to reduce rate of synthesis of that product concentration ofendproduct signal that quotfeedbackquot inhibits rst committed step of pathway 04 mM CTP Rate of N carbamoylaspartate formation gt 1 o 20 AspartatEL mM Berg et al Fig 043 LEC 15 Enzymes Regulation 1 BIOC 460 Spring 2008 Heterotropic Effects in ATCase ATP is an allosteric activator of ATCase preferentially binds to R state shifts RT equilibrium toward R state which binds Asp more tightly so VO vs 8 curve shifts toward LEFT as shown in blue Competes with CTP for binding the regulatorynucleotidebinding site on regulatory subunits W ST 2mMATP E 5 o m 35 Tu39 E m E 6 EV M Y 2 1O 20 Aspartate mM Berg et al Fig 1014 Heterotropic Effects in ATCase continued ATP activates ATCase and thus leads to more pyrimidine biosynthesis ATP is a Qurine nucleotide not related to the pyrimidine biosynthetic pathway Why would ATP be an allosteric activator of ATCase ATP is used to quotstorequot metabolic energy in the cell High concentration of ATP is an intracellular indicator that the cell is energyrich very quothappyquot metabolically High ATP concentration thus quottellsquot the cell there are lots of purine nucleotides available so more pyrimidine nucleotides are needed to keep nucleotide pool balanced for nucleic acid biosynthesis and cell is in great shape metabolically and wants to replicate its DNA and divide high concentration of nucleotides is needed for cell division High ATP thus can quotoverridequot inhibitory signal of high CTP and activate ATCase ATP binds to the same nucleotide binding site on the regulatory subunits that CTP binds to if CTP binds equilibrium shifts toward T state if ATP binds equilibrium shifts toward R state lsozymes lsoenzymes Multiple forms of enzyme that catalyze same reaction Different amino acid sequences products of different genes Expressed in different tissues or organelles at different stages of development to meet different metabolicregulatory criteria Different kinetic parameters like Km andor different allosteric regulation with physiological consequences Example hexokinase in muscle vs glucokinase in liver muscle function contraction breaks down glucose for energy gets glucose from blood 1 major liver function maintenance of blood glucose at 45 mM liver takes up and stores excess glucose or makes more glucose and exports it as needed Function of hexokinaseglucokinase glucose entering cells from blood is phosphorylated trapping it inside charged compound can t get back out Hexokinase muscle low Km for glucose 01 mM so working at Vmax since cellular glucose 2 5 mM inhibited by product glucose 6phosphate if G6P is building up muscle won t take more in from blood Hexokinase IV Glucokinase liver high Km for glucose 10 mM so activity regulated by blood glucose not inhibited by product G6P LEC 15 Enzymes Regulation 1 lsozymes of hexokinase different metabolic roles Kinetic properties of hexokinase muscle and hexokinase lV glucokinase liver fit different metabolic needs in liver and muscle Blood glucose 45 mM Hexokinase muscle Km 01 mM already operating near Vmax when blood glucose increases above 5 mM so little change 0 Hexokinase l Glucokinase liver Km 10 mM regulated directly by changes in conc of blood glucose VO vs glucose changing steeply in glucose range below Km Hexokinase IV glucokinase Relative enzyme activity Glucokinase has other kinds of regulation too o 5 1o 15 20 Nelson amp Cox Lehninger Principles Glucose concentration mM ofBIbchemistry 4th ed Fig 1516
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