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by: Jaden Stiedemann


Jaden Stiedemann
Rice University
GPA 3.78


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This 12 page Class Notes was uploaded by Jaden Stiedemann on Monday October 19, 2015. The Class Notes belongs to BIOS 301 at Rice University taught by Staff in Fall. Since its upload, it has received 33 views. For similar materials see /class/225029/bios-301-rice-university in Biological Sciences at Rice University.

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Date Created: 10/19/15
BIOS 302 Lecture 12 Olson Anaplerotic Pathways Carboxylations and Gluconeogenesis L FOB14 pp 543 549 LPOB15pp 571582 reversible steps in glycolysis ozcc HCOg 0P0 poi oc CH3 pyruvate Alternative fates of PEP and pyruvate in mammalian systems see Lec6 and Lec7 ADP I ll ATP quot Passes through blood vessels back C01 GD CH1 l39 to the liver or glucone genesis Glucuneugenesis PEP NADHH NAD 39 and secretion as glucose The Cori quot C cle back to C3 with GT1 PEPCK COz y catalytic use oi C02 7 OC HO IH cytoplasmic and C 0239 C H3 LDH CH3 mitochondrial PEPCK 0C pyruvate Liladate cytoplasm C Hz rimary skeletal 3910 7 i muscle product 2 oxaloacetate 39 39 mitochondrial malateDH NADHIF II membranes 302 NADquot quot i All aplerotic pathways i MItDChondrlal HO39C H matrix I Filling up TCA cycle with C H I intermediates generating pyruvate 39 CO 39 z from amino acids and Lmalate gluconeogensis CO ATP C02 0C z CoASH 1 5 ADP Pi CH3 pyruvate 5 oxidation of 3 amino pyruvate NAD fatty acids 39139 ands Coz39 carboxylase H 9 i transaminases OC biotin NADH39 C tcssc A I o 39l pyrid oxalPi EH1 C3 to C4 3 C AcetleoA I oxaloacetate malateDH CoASH l NADHH 7 C0239 C02 NAD OC 017 HOCH sz Hz H O C CO 39 C H 2 z 30 m The TCA cycle EH2 C027 oxa oace 6 complete oxidation I t c 0 C02 L39malaw malic enzyme 0 z N AD Mn2 C4 10 C3 Cytoplasmic mitochondrial or chloroplast enyzme N AD Pva A Introduction there is a need for anaplerotic pathways to keep the levels of TCA intermediates high enough for rapid catalysis 1 There is a continual removal of intermediates by anabolic processes Le fatty acid and amino acid syntheses and thus there is a need to C3 gtC4 transformations to make TCA cycle acids 2 Oxidation of amino acids requires a continuous supply of acetylSCoA units and there is a need for C4 gtC3 transformations to make pyruvate and then acetylSCoA not for Bios 301 3 Carbohydrate oxidation needs to be quotprimedquot by a build up TCA cycle intermediates for efficient catalysis C3 gtC4 4 Fatty acid oxidation via acetleoA also requires carbohydrates or amino acids to quotfillupquot the TCA with intermediates 6 g citrate helps the metabolism of fats 7 quotgrapefruit dietquot strategy not for Bios 301 B Major Anaplerotic Pathways C3 ltgt C4 interconverstions 1 Transaminase reactions conversion of alanine aspartate and glutamate into pyruvate oxaloacetate and ocketoglutarate respectively These reactions are also the first steps in either gluconeogenesis or lipogenesis from amino acids see Lecture 3 pp 6 not for Bios 301 The malic enzyme generation of pyruvate and thus acetleoA from C4 TCA cycle intermediates in mitochondria production of NADPH in cell cytoplasm from amino acids for lipid biosynthesis LPOB 21 and release of C02 for the Calvin cycle in bundle sheath cells of C4 plants LPOB 20 pp 769700 Fig Equot 20 23 NADP NADPHF CO2 W2 COZ39 HO H OClt co2 EH2 malic enzyme CH3 COZ CA to c3 pyruvate Lmalate a This enzyme is required for lipogenesis fatty acid synthesis from most amino acids LPOB21Y0u don 39t need to know this pathway for Bi0s301 1 pyruvate CO2 2 aspartates acetylSCoA 391ansaminati0n Bs cytoplasmic fatty acid syn esis 2 oxaloacetates A malateDH 3939 cytoplasm or NADHHJr I mitochondira citrate 39 CO NADJr 1 malate TCA cycle Oxidation of amino acids in TCA cycle and fatty acid syntheisis malic enzyme from proteins N ADpJr cytoplasm or the simultaneous generation oI NADPH b The mechanism of the malic enzyme involves intermediate decarboxylation of a Sketo acid It is analogous to the isocitrate dehydrogenation reactions which also involves oxidation of a SOH group followed by Mn2 catalyzed decarboxylation see Lec4 p 5 2 Sketo acid decarboxylation NADP NADPHH COz39 co H CO 39 2 r n 2 co2 co2 HO f H ant OClt Mn2 LL OC EH2 malic enzyme CH2 CH CH3 CO C4 to C3 I 2 pymvate 2 A0 Lm alate O O39 3 Formation of phosphoenol pyruvate from TCA cycle intermediates PEP carboxykinase GTP oxaloacetate decarboxylase LPOB 14 p 546 a Animal PEPCK found in mitochondrion liver and cytoplasm muscle for gluconeogenesis Icoz39 Mg2 Mn2 3902C OP03 Probable mechanism OC GTP CO2 GDP decarboxylation followed by CIHz PEPCK CH2 phosphorylation of the resulting coz39 enol pyruvate ie decarboxylation driven reversal of the pyruvate PNPPGd kinase reaction see Lec6 p 45 I I h drol sis of GTP to GDP with the c t y y anr 5 ICHZ ant CgCCHZ CO2 CO2 cof The reaction is driven by the phosphate being put on enol pyruvate C O O b The C4 tropical plant enzyme PEP carboxylase carries out the opposite reaction CO 02 130 Oc 2 72f5a327 32 250 by C HCO PEP carboyxlase g 3 CH2 mesophyll cells of C4 plants and is CH2 1 I30 part of the H atch Slack pathway P i HO 2 for C02 assimilation LPOB20 Fig H y 20 23 pp 769770 The reaction is C02 2 302 09 0 driven by the hydrolysis of the Blcquot O KQ J Mn2 phosphoenol quothigh energy39ibonal CH I 0 You don 391 need to know this 2 H H reaction for Bios 301 4 ATP dependent pyruvate carboxylase the quotprimingquot step in carbohydrate oxidation and the rst step in gluconeogenesis from lactate see pp 1 and 7 and LPOB l4 p 545546 coz CO 2 2 I I 2 Mg M K OC ADP Pi OC ATP HCO339 CH CH3 I 2 pyruvate C02 Requires acetleoA as an allosteiic activator C Pyruvate carboxylase the classical example of a biotin containing carboxylase enzyme LPOB 716 pp 618620 1 Properties of the liver enzyme a MW S 650000 large complex b 4 subunits with MW S 150000 Daltons c Each subunit binds 4 biotins 4 Mn 4 pyruvate substrate molecules and 4 acetleoA allosteric effector molecules 2 Properties of the cofactor biotin vitamin H It generates quotactivatedquot C02 complexes for carboxylation of carbon atoms Ct to keto groups the opposite of the Sketo acid decarboxylation reaction described above for the malic enzyme Structure of biotin a fused ureido and tetrahydrothiophene rings plus an nvaleric acid side chain which is attached to a lysine side chain by an amide linkage similar to lipoamide arm for PDH Lecture 7 p 6 LPOB Fig 1418 site of HCOS39 attachment 0 ll Rings are quotkinkedquot quotquotquotquotquot 39 backwards Hi39TH H H mu VW CN 4 14A backbone Biotin V Biocytin 3 Pyruvate carboxylation is carried out by a twostep mechanism which is similar for all biotin catalyzed reactions The steps are 1 activation of HC0339 by hydrolysis of ATP and adduct formation with biotin 2 transcarboxylation by transfer of C02 from the biotin to the acceptor molecule which in this case is pyruvate a First step formation of carboxybiotin Acetyl CoA 0 Acetyl CoA is required but not Mn2 Proof i Exchange reactions that work and require bicarbonate HC0339 Ebiotin Arm1g HCO339 EbiotinNCOZ ADP Pi 14 EacetleoA 14 E Bicarbonate is required ATP ADP C HCO3 ATP C for these exchanges E ace 1C0A ATP PiJr HCO3 ATP32P ii EbiotinNMCOz can be isolated from a mixture of the enzyme H14C0339 and ATP Mechanism at the HCOg39 activation site S Sulfur atom of the thiophene 39 ring serves as an quotanchorquot for H the biotin molecule A Activation of HCO3 by formation of a carbonic acidphosphoric acid anhydride PP Ad O NCNH A m 3911 a Mg OC oD quot 0 Formation of active quotC01quot or carbamate H on biotinN atom O PP Ad a s o x quotActive C01quot HN NC H Mgz P 0 H0gt 039 O AH NuC I 390 o 0 Y H II 0 a l Cc1fz quotActiveCOzquot HNCNC 11n1 or 0H2 2 transcarboxylation site an binds here 1 HCO activation site AcetleoA binds here Two physically distinct active sites b Second step transcarboxylation from biotin to pyruvate COZ39 Emu c02 E biotinNCOZ OC z Ebiotin OC CH3 but not CH acetyl CoA l 2 pyruvate CO2 oxaloacetate Proof i Exchange reaction coz39 CCOZ39 EM CCOZ39 z Oc O O 1 CH2 CH2 14CH3 coz39 coz39 Pymvate cold oxaloacetate oxaloacetate 11 C4 labeled oxaloacetate covalently labels the enzyme making Ebiotin14C02 Mechanism at the transcarboxylation site 1 H Lnnksfnrmzlly likeuczrbaninn 0 0 atmck o eczrbnxylatinn c 0239 o c Enxyme bound bincyLin H c H c H CH2 N N N s 4 Similar biotin carboxylase enzymes a Acetyl CoA carboxylase 7 1St step in fatty acid biosynthesis so A M 2 M 2 SCOA o I 0 g n 0 ADP P 39 C ATP Hco3 blotm CH2 CH3 I acetleoA CO2 Requires citrate malOIIYICOA as an allosteiic activator b Propionyl CoA carboxylase 7 required for the complete oxidation of odd chain fatty acids so A 2 2 SCOA O O N Mg Mn Oq ADP Pi C ATP Hoo3 gtbi0 n CHCOZ ltin I CH3 CH3 propionylcoA methymalonleoA m y orsxr m m we mm m m r quot We 48 A A V MW anvx m m lymlysn mammal s Hmwlmglvmmu Imlnmy u rmamn mu msmmemuwmm m m 0mm m r Mmquot L m M mmquot mm M W m quot35 so 7 m We rmr l w wnl s SecrenunufglucusebyGr Whl xsfu s Rwasxble steps m glyculysxs Heep m PEP Hurmunal andallusten regulauun ufglycugen metabuhsmEm53Z thuspha39zse eh und unlym uver ee11s andallusten regulauunuftheFFKrl enema 251wlesz may FFKFBFr Fmtankmase A eAMF glumguns epmephnne Allusten and hurmunal regulauun ufpyrumle kmaseFK Bypass ufpyrumtekmase PK step by arbuxylauun ufpymvale m uxaluacmzle pyruvate arbuxylase and remaan ufFEF frunquot uxaluacmzte PEP carbuxykmase Ifs39amng rmmlaewemen cycle pm a mzed m uxaluacmzle m pruduce me NADH needed m reduce glycaate m me GAFDH step l The mechanistic strategy for irreversibly converting pyruvate to PEP is to drive the reaction to completion by hydrolyzing GTP and ATP and using bicarbonate catalytically blue reactions below Allosteric effectors activator inhibitor ADP Deactivated by PKA ATP Activated by GPP ATP acetleoA lactate fatty acids pyruvate kinase 02C OPO3 C02 reversible steps 0C The TCA cycle complete in glycolysis CH2 CH oxidation to C02 HCO 39 3 I tr 1 PEP CO2 pyruvate resplra ory con 0 02 Alanine OC GDP CH2 ATP pERcarboxykinase GTP OZV ADPYPi pyruvate carboxylase oxaloacetate 0AA t xcetleoA ireqllir ed 2 The real pathways interconnecting the mitochondrial and cytoplasmic steps depend on the starting substrate for glucose synthesis see page 1 and LPOB Fig 1419 a When starting with ocketo acids normally amino acids oxaloacetate is converted to malate which is transported into the cytoplasm and then oxidized back to oxaloacetate to produce the NADH needed for reversing glycolysis b When starting with lactate NADH is generated in the cytoplasm by LDH activity Then the pyruvate generated is transported into the mitochondrion carboxylated to oxaloacetate converted directly into PEP by mitochondrial PEPCK and then PEP is transported out of the mitochondrion 3 The other cytosolic control points in glucose synthesis after PEP is generated from Llactate a Hexose monophosphate formation and hormonal regulation via F26Pz and PFK2FBP2 see Lec5 pp 1 2 LPOB pp 576 582 FBP phosphataserl FBPrl ATP fatty acids AMP ADP NH 3 26P2 PEP fructose7167P2 fru osei iPi ADP ATP Amdtme 1 AMP ADP NIL F6PF26Pz Phosphofructokinaser 1 PFKV 1 b Glucose secretion glycogen metabolism will be covered in Bios 302 Glucosei iP phosphatase iver only 1 Cell blood glucose64 glucose glucose 39 ADP ATP l f Hexokinase I 11 HI muscle brain 7 low KM Hexokinase IV liver 7 high KM regulatory I protein and nucleus localization E Regulation of blood sugar levels by glucagoninsulin and removal of lactate by the Cori Cycle Blood sugar levels LPOB Fig 2323 Blood glucose my 100 mL Diabetes if prolonged Transienlly high after a meal rich in sugars Normal 45 mM range Insulin secreted Hormonal Regulation LPOB Figs 2326 2327 Blutft Low g1 ut i I Subtle neurological signs hunger 1 1 I Release ofglucagon epinephrine cortisol legen z u Sweating trembling I Glucose Glucagon Lethargy pymmm Glucose Convulsmns coma C02 Muscle Permanent brain damage if prolonged Death Insulin stimulntes glucose uptake and Consumption High blood Muscle AT P produced by Ha i Lactate removal LPOB Fig 2318 glyt39nlysis inquot rapid contraction 7 I Pancreas Lac arclt7 Glycogen A blond ATP quot muquot 1 V Blow Blood lot late glucose Glycogen Glucose Pyruvato r LattateT Glucose Liver ATP llumgnn stimulates gluctme synthesis and export nf glucose gllit onengeneaial during recovery Liver ATP used in synthesis a Stimulation of glucose uptake and metabolism occurs by the binding of insulin to a transmembrane receptor which is a tyrosinespeci c kinase see LPOB3 p 97LPOB12 p 429 433 LPOB 23 p 887 902908 UH Structure of bovine insulin HIV 01 LL M llLL TABLE 23 3 A chain A gt gt 1 r f E z A v 391 I 2 Z 1 gt f 1 f lt 7 gt lt 7 3 1 39 lt L gt r Z v 7 c lt u B Chain Effects of Insulin on Blood Glucose Storage as Triacylglycerols and Glycogen Metabolic effect 392 a 7 gt t A 2 3 lt 5 7 s 1 r v 394 z A 1r 7 39 I v r A t 2 lt1 7 2 394 1 m 39I v v z 3 Uptake of Glucose by Cells and Target enzyme T Glucose uptake muscle adipose T Glucose uptake liver T Glycogen synthesis liver muscle i Glycogen breakdown liver muscle T Glycolysis acetyICoA production liver muscle T Fatty acid synthesis liver T Triacylglycerol synthesis adipose tissue T Glucose transporter GLUT4 T Glucokinase increased expression T Glycogen synthase l Glycogen phosphorylase T PFKl by T PFK2 T Pyruvate dehydrogenase complex T AcetylCoA carboxylase T Lipoprotein lipase l Insulin receptor LPOB Fig 126 2 Insulin activated mobilization of glucose transporters LPOB Box 1112 Insulin linimrl to receptor sites nsuin Receptor When insulin interacts with its receptor vesicles A tyrosine Speci c move to surlace and fuse With the plasma k membrane increasing the number of glucose 6 Extracellular protem mase transporters in the plasma membrane 39 V Insulin 39 When insulin level drops space membrane V components a glucose transporters are Pmtein kinases 39 removed from the plasma 39 membrane by endocytosis l P W l ormmg smell vesmles r l L IRS1 C amp Glucose Insulin receer Plasma membrane W E o T kiuusc amp drumnus Autnphos pllurylulion Carlxlxyllenlunnl Rite Glucose transporters Target lnrnnins stored within cell in protein Sl membrane vesicles m T T ADP see LPOB Figs 12 s and M R Table 233 Q lnsulin TSCCPIUI subsiraLcl 39 Intracellular 3 insulin egre s Patches of the endosome enriched for The smaller 311105 transporters bud olfto become vesicles use with vesxcles ready to return to the W surface when insulin levels rise again larger mdmome transporter vn Tyrosine Cylosol 2 Glucagon regulation of glucose metabolism and stimulation of gluconeogenesis G ProteincAlVlP second messenger receptor analogous to Badrenergic receptor for epinephrine Bios302 LPOB Chapter 13 pp 449455 Glucagon primary sequence HSEGTFTSDYSKYLDSRRAQDFVQWLMNT TABLE 23 4 Effects of Glucagon on Blood Glucose Production and Release of Glucose by the Liver Metabolic effect Effect on glucose metabolism Target enzyme T Glycogen breakdown liver Glycogen gt glucose T Glycogen phospliorylase l Glycogen synthesis liver Less glucose stored as glycogen l Glycogen synthase l Glycolysls liver 4 Less glucose used as fuel in liver gtl PFK 1 T Gluconeogenesis liver Amino acids T FBPase2 Glycerol gt glucose gti Pyruvaie kinase Oxaloacetate l T PEP carboxykinase T Fatty acid mobilization adipose tissue Less glucose used as fuel by liver muscle T Triacylglycerol lipase Perilipin pliosphorylation T Ketogenesis Provides alternative to glucose as T Acetyl CoA carboxylase energy source for brain 1 Effects of glucagon binding to liver membrane receptor are mediated through cAMP as a second messenger Glucagon binding to gt GSuproteinGTP membrane receptor 2Pi Adenylale Cyclase I membrane bound ppi NH2 NH2 N N if 0 o o ll II N N H OD oPO lt l hr 00 2 0 0 CH2 0 l quot J ogp Mg2 39 OH 2 cyclic nucleotide 39 ATPWIQ OH OH phosphodiesterase CAMP soluble cytosol 39 Inhibited by methy xanthines Activation of protein caffeine theophylline Kinase NPKA lO 2 Mechanism for activation of adenylate cyclase and Protein Kinase A D Glucagon binds to its specific serpentine receptor on hepatocyte membranes The occupier receptor causes replacement of the GDP bound to G5 by GTP activating 63 See LPOB Figs 1212 and 1213 Key effect of cAMP is to turn on Protein Kinase A old name general protein kinase which phosphorylates a number of key regulatory enzymes in gluconeogenesis and glycogen metabolism Inactive PKA Regulatory subunits GTP GDP 1 39 empty CAMP sites l Catalytic subunits substrate binding sites blocked by autiziirihibitoryr domains ofR subunits Adenylyl cyclase catalyzes the formation of CAMP GB a subunit moves to adenylyl cyclase and activates it cAMP l l 39 i ij ll39 HIll till I SKA 395 getlvat ed iiimplioiiiuslei39nsr y cAM Regulatory subunits aiiminhibitory domains buried US CD Pliosphorylation of CAMP is degraded cellular proteins by reversing the PKA causes the activation of PKA l I cellular response to Active pKA epinephrine and glucagon Catalytic subunits C ripen sulistmte binding sites 3 InactivationActivation of PFK2FBP2 activity by PKA activity LPOB pp 731 733 gluconeogenesis Fructose26P2 CH20P03OPOS allosteric effector to O activate PFK1 and inhibit FBP1 ATP DP PFK2FBP2 regulatory 39 CHZOPOs enzyme with CHon 0 both activities V Activity regulated by PKA CAMP glucagon which AG 167 kJmol CHZOH turns quotoftquot F26 P2 formation Keq 850 Fructose6 P 1quot ve 39 CAMP luca one ine hrine FBP1 PFK 1 All Pi V OH 9 g p p activates 1 1 mac Iva es 39539 f ATP V 0 r inac ive CHZOP03 AG 17392 kJmOI General protein 39 Protein Kinase A o Ke 1000 q phosphatase insulin stimulated 39quotact39Ve ADP CH20P03 Fructose16 P2 i glycolysis ac ve 4 Activities of PFKl and FBPl in the presence and absence of F26P2 Muscle and brain enzymes are 39 M a mm min pmeemeaew 39onquot at low ATP 3 005 m u fnrglucnrmgmus Mumlmlnwnt39WIdwny inTe rmedietelAYPicauses 26er memequot sigmmdaikinetms K 0 WW Otf eth h mm M amasr aciwny 9 i I 39 activity new W W mm m 1 n 2 n 3 Fhosphofructokinase1 PFK1 crystal structure Allosteric site for and t e enzyme activated this effect overcomes lack of F26Pz If F 26Pz is the effector its ieveis are regulated by phosphorylation of the turn is regutated by the hormone Gupmt in receptorcAMPPKA system glucagonIiver


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