Review Sheet for BIOC 460 at UA
Review Sheet for BIOC 460 at UA
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Date Created: 02/06/15
BIOC 460 Summer 2009 Enzymes Regulation 1 Allosteric Regulation and lsozymes Reading Berg Tymoczko amp Stryer 6th ed Chapter 10 pp 275283 T state inactive ATCase R state active aspartate transcarbamoylase allosteric regulation Enzymes Regulation 1 BIOC 460 Summer 2009 Key Concepts Amounts of many key enzymes are regulated at the level of control of transcription mRNA processing andor translation mechanisms covered in BIOC 411 or BIOC 461 or destruction proteolytic degradation of oldunwanted enzymes Activities of many key enzymes are regulated in cells based on metabolic needsconditions in vivo Regulation of enzyme activity can increase or decrease substrate binding af nity andor kcat 5 ways to regulate protein activity including enzyme activity allosteric control multiple forms of enzymes isozymes interaction with regulatory proteins reversible covalent modification irreversible covalent modification including proteolytic activation 91900 Enzymes Regulation 1 BIOC 460 Summer 2009 Key Concepts continued 1 Allosteric control conformational changes 2 conformations in equilibrium quotRquot more active amp quotTquot less active allosteric activators positive effectorsmodulators allosteric inhibitors negative effectorsmodulators often feedback inhibitors product of pathway inhibits first committed step in pathway Allosterically regulated enzymes always multisubunit Aspartate transcarbamoylase ATCase as an example homotropic effector activator substrate aspartate heterotropic effectors activator ATP inhibitor CTP 2 lsozymes Multiple forms of an enzyme that catalyze the same reaction Different kinetic parameters like Km andor different allosteric regulation with physiological consequences Hexokinase different forms in liver vs muscle reflect the different roles of those tissues in the body Enzymes Regulation 1 BIOC 460 Summer 2009 Learning Objectives Terminology some are review quaternary structure multimeric protein homopolymeric protein heteropolymeric protein ligand binding site fractional saturation feedback inhibition cooperativity cooperative binding allosteric homotropic effectorregulator heterotropic effectorregulator allosten39c activator positive heterotropic effectorregulator allosteric inhibitor negative heterotropic effectorregulator protomer prosthetic group isozyme Briefly explain the allosteric regulation of ATCase 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 V0 vs 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 Vo vs aspartate curves with no heterotropic regulators present with an allosteric inhibitor present and with an allosteric activator present Explain the biological usefulness of isozymes and discuss the example of muscle hexokinase vs liver glucokinase in terms of difference in function of the tissues Enzymes Regulation 1 BIOC 460 Summer 2009 Regulatory enzymes In general Catalyze essentially irreversible metabolic reactions AG large neg Catalyze the first committed step in a metabolic pathway Regulation of such steps permits ef cient regulation of flux of metabolites through just that pathway quotCommitted stepquot This step commits a metabolite small molecule down pathway to endproduct No other branches lead to different endproducts that need to be regulated separately FIRST committed step most ef cient step for regulation ofthe rate typically is the slowest step in pathway controlling quot oW39 ofmatter to endproduct whose concentration you want to regulate Feedback inhibition endproduct acts as an allosterie inhibitor of enzyme camlyzillg FIRST COMMII39I39ED STEP in that pathway t E5 E1 E1 E3 E 4 F endproduct A A B a C 4 D 15gt Q 4 R 4 S endproduct 8 E7 Es 1 Enzymes Regulation 1 BIOC 460 Summer 2009 5 principal ways proteinenzyme activity is regulated 1 Allosteric control Regulate binding affinity for ligands andor of catalytic activity by conformational changes caused by binding of the same or other ligands at other sites 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 Enzymes Regulation 1 BIOC 460 Summer 2009 5 principal ways proteinenzyme activity is regulated 4 Reversible covalent modi cation 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 Enzymes Regulation 1 BIOC 460 Summer 2009 Allosteric Regulation Multisubunit enzymes more than one active site per enzyme Regulation of binding af nity for ligands like substrates andor catalytic activity kcat 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 subunits 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 Enzymes Regulation 1 BIOC 460 Summer 2009 Allosteric regulation permits very rapid cycling of enzyme between more active and less active conformations Allosteric activators gt increase activity Homotropic effector substrate itself Heterotropic effectors eg Metabolite product from a reaction upstream feedahead activation Other metabolites ligands from other pathways that act as indicators of cellular metabolic need Allosteric inhibitors gt decrease activity Heterotropic effectorsmodulators Endproduct of whole pathway feedback inhibition another ligand that acts as indicator that cell needs less of that pathway s product Enzymes Regulation 1 BIOC 460 Summer 2009 Homoallostery Allosteric enzymes have Sigmoidal Vo vs S plots look familiar 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 Homoallostery effect is due to the Substrate alone Noncooperative Loo Enzyme 075 gt 050 Allosteric Enzyme 025 000 0 25 50 75 100 S Enzymes Regulation 1 B OCMSO Summer 2009 Heteroallostery n by heterotropic effectors nonSubstrate e ac ation favors R state or negative inhl tIon favors T state Heterotropic effectors bind to differentsite from active site 100 Positive effector 075 Basal rate 050 Negative effector 025 Physiological S 000 0 25 50 75 100 Enzymes Regu atwon 1 BlOC 460 SummerZOOQ Aspartate transcarb amoylase Pyrinidim nudeo de biosyntlmsis quotATC eedback inhibition of bacterial 359 asparfate transmrbamoylzse first committed step in the pathway for hiosynthesis of Aslmme pyrimldine nucleotides ATCase Nucleotides P 05 3 compounds whose 3 covalently Pi linked components are Nmrbmnoylaspamm heterocyclic quotbasequot A G C orTin DNAusuallyAGC l or U in RNA l sugar deoxyribose in DNA J immh39t39aquot ribose in RNA 3 phosphate l building blocks of nucleic acids UMP other major roles x 3 coenzymes energy storage compounds UT regulators of enzyme activity i Enzymes Regulationl B OCMSO SummerZOOQ ATCase reaction Condensation ofAsp carbamoyl phosphate gt carbamoyl aspartale P NH H 06 v upog ou Curhnmuyl Aspunnve Ntuvhumoyluspurlnle phosph e Hz 4 N o endproduct ofpathway CTP H0 H HE S 3 3 F B 394 tyridina lriplmsplmle cm Enzymes Regu atwonW BiOC 460 Summer 2009 ATCaSe MC H 1 mot NH nzu H mm Inund subslrnles PALA a bisubstrate analog for ATCase Bmds to acme sue ofand stabmzes R state acme conformation of Can t react to form products Reunion inielmedinl 0 Note resemblance of PALA o to reaction intermediate f u quotE II M 3900 N n NI39llospllnnluaiylluspnrlnu mu Eevg a ai Fig m7 Enzymes Reguiauon 1 14 BiOC 460 Summer 2009 Homoallostery in ATCase In absence of any substrate or regulators ATCase RT equl rium favors T state by a factor of about 200 Tn I R0 200 T state has a very HIGH Km for Asp R state predominates at high 8 has a much LOWER Km forAsp i R state low Km formation gt T state hi h Km 9 Rate of Narbamoylaspartate 1 0 20 30 4D Aspartate mM ATCase binds the substrate aspariate cooperatively sigmoidal kinetics Enzymes Reguiationi i5 BIOC 460 Summer 2009 As Asp increases enzyme shi s from T to R 1Activity increases steeply 2 apparent Km decreases giving the sigmoid plot of VEl vs Asp Rate of Ncarbamoylaspartate Look familiar Berg et al rig 1040 formation gt ATCase Substrate Binding cooperative substrate binding mixture of R and T states Equilibrium at very low 8 lies far toward T conformation TR 2001 As Asp binds T ltgt R equilibrium shi ed to right ATCase activity Rstate curve Tstate curve Aspartate gt Enzymes Regulation 1 16 Bloc 46D Summer ZEIEIB Structural basis for allosteric regulation in ATCase Quaternary structure subunit structure of ATCase 7 l2 subunits 6 catalytic cnains total arranged in 2 c3 catalytic trirners blue 6 regulatory chalnstotal arranged in 3 r2 regulatory dimers red Catalytic trirners c3 catalyze reaction in absence or regulatory dimers For isolated catalytic trirners c3 substrate Asp binding is NOT cooperatiye no communication between catalytic subunits in c3 Feedback innibitor CTP nas no errect on actiyity of isolated catalytic trimers not surprising e CTP binds to regulatory subunits Regulatory subunits bind heterotropic effectors CTP feedback inhibitor and ATP allosteric activator Sllyel dlh ed Enzymes Regulatlunl l7 B OC 460 SummerZOOQ Another view of ATCase structure Regulamry dimer A Zinc domaln w 1 chain Calalyxic 3 winner negnanny R um dimev 5 0W dimer Sula View Regulatory dimer Catalytic lrimer Berg EtaLng mra Enzymes Regu atwonW 18 Bloc 46D Summer ZEIEIB ATCase active sites at interfaces between catalytic subunits 3 active sites per catalytic trimer PALA bisubstrate analog binds very tightly interacting with residues on both sides of subunit interraces Elulyxi subunil Arg 229 Enzymes Regulatmnl ElOC 46D summer 2mg T state tense form more compact less active 6A lo39 T slllle Quaternary structural changes T gt R in ATCase R sme relaxed ronn expanded more active favored by PALA binding PALA a bisubstrate analog and by ATP binding R sill am el al rig l ra Emymes Regulatlunl 2n BiOC 460 Summer 2009 T state less active stabilized by CTP binding CTP is a feedback innibiton tne endproduct of tne pathway of pyrimidi e nueieotide biosy thesis CTP bind to reguiatory Subunit and iocks enzyme in T state Eevgeiai Fig i r T slnle CTP T silie Enzymes Reguiatiori i 21 BiOC 460 Summer 2009 Quaternary structural changes T gt R in ATCase In absence of any substrate or regulators ATCase RT equilibrium ravors T state by a factor of about 200 in I Rn 2oo 39l slnle less anin R slim more active Favored by CTP binding Favured by substrate binding aergetai Fig WEHZ Enzymes Reguiatron i 22 BIOC 460 Summer 2009 Heterotropic Effects in ATCase Heterotropic ligands bind to the regulatory subunits of ATCase CTP endproduct ofwhole pathway allosteric inhibitor of ATCase binds preferentially to T state of whole ATCase thus decreasing binding af nity for Asp substrate at active sites on catalytic subunits Lower af nity for Asp means apparent Km increases so at any given Asp concentration VB is decreased This is the essence of FEEDBACK INHIBITION the endproduct of pathway CTP signaling back and slowing down the rst committed step 04 mM CTP Rate of Ncarbamoylaspartate formation gt 10 20 Aspartate mM Enzymes Regulation 1 23 BIOC 460 Summer 2009 Heterotropic Effects in ATCase ATP a purine is an allosteric activatorofATCase preferentially binds to R state shi s RT equilibrium toward R state which binds Asp more tightly so VB vs S curve shi s toward LEFT as shovm in blue Competes with CTP for binding the regulatorynucleotidebinding site on regulatory subunits m u QT 2mMATP N u 5 o N sin gt5 5 E n E 5 3 IE 739 z 10 20 Aspartate mM Berg etal Fig 1044 Enzymes Regulation 1 BIOC 460 Summer 2009 Heterotropic Effects in ATCase continued ATP activates ATCase and thus leads to more pyrimidine biosynthesis ATP is a purine 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 an indicator that the cell is energyrich 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 Enzymes Regulation 1 25 BIOC 460 Summer 2009 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 Both enzymes phosphorylate glucose inside cells using ATP trapping it inside Hexokinase l 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 muscle contraction requires a lot of energy derived from blood glucose Glucokinase liver high Km for glucose 10 mM so activity regulated by blood glucose not inhibited by product G6P 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 Enzymes Regulation 1 BIOC 460 Summer 2009 Blood glucose 45 mM Hexokinase muscle Km 01 mM already operating near VmaX when blood glucose increases above 5 mM so little change Glucokinase liver Km 10 mM regulated directly by changes in conc of blood glucose Vo vs glucose changing steeply in glucose range below Km Relative enzyme activity Dr Tischlerwill talk about this in more detail later Nelson amp Cox Lehninger Principles of Biochemistry 4th ed Fig 1516 10 lsozymes of hexokinase different metabolic roles Hexokinase I Hexokinase IV glucokinase I 5 1o 15 20 Glucose concentration mM Enzymes Regulation 1 27
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