Eukaryotic Exam 2

module 5

evolution of aerobic respiration

• in the first 2 billion years the atmosphere was made of H2, NH3, and H2O; organisms were anaerobes so they could only do glycolysis and fermentation

• about 2.7 billion years ago was the appearance of cyanobacteria using light energy to split water and release O2 into the atmosphere

• species evolved that were not only protected from the damaging effects of O2, but possessed metabolic pathways to utilize O2

• the O2-dependent prokaryotes and eukaryotes living today evolved from earlier aerobes

• in eukaryotes, the utilization of O2 takes place in mitochondrion, which may have evolved from an ancient aerobic bacterium that took up residence inside the cytoplasm of an anaerobic host cell

mitochondria characteristics

• big enough to be seen under a light microscope

• are surrounded by a semipermeable membrane

  • double membrane

• have very different overall structure; can be sausage-shaped to highly branched, interconnected tubular network

• can fuse with one another or split in two

dynamic structure of mitochondria

• mitochondria fuse with one another or split in two

• the balance between fusion and fission is likely a major determinant of mitochondrial number, length, and degree of interconnection

• photo is of mouse fibroblasts with a fluorescent tagged mitochondrial protein; shows how mitochondria can fuse and split

mitochondrial fission

• mitochondrial fission is apparently induced by contact with thin tubules from the ER, which can encircle the mitochondrion like a noose

• these ER tubules appear to initiate constriction, which is then completed through the action of soluble proteins that are recruited to the outer surface of the mitochondrion from the cytosol

mitochondria structural components

• outer mitochondrial membrane

• inner mitochondrial membrane, including inner boundary membrane and internal cristal membranes (crystae); joined to one another by narrow connections, but they are functionally distinct

  • inner membrane and cristal membranes are continuous

• intermembrane space

• matrix

outer membrane

• approx. 50% lipid by weight and a mixture of enzymes involved in diverse activities, like oxidation of epinephrine, degradation of Trp, and elongation of fatty acids

• homologous to an outer membrane present as part of the cell wall of certain bacterial cells

  • both contain porins; integral proteins that have a relatively large internal channel surrounded by a barrel of β strands

• the porins of the outer membrane can undergo reversible closure; when they are wide open, the outer membrane is freely permeable to molecules such as ATP, NAD and coenzyme A

inner membrane

• 100 different polypeptides and a very high protein/lipid ratio (>3":1)

  • the composition and organization of the IM are the key to the bioenergetic activities of the organelle

• rich in cardiolipin; characteristic of bacterial plasma membranes, from which the inner membrane presumably evolved

• highly impermeable; all molecules and ions require special transporters to enter the matrix

membranes of gram-negative bacteria

• shows to membrane layers; outer and plasma (?)

  • homologous to the membrane of mitochondria

• shows porins: a β-sheet barrel that forms an opening for passage of moderate-sized molecules (metabolites)

mitochondrial matrix

• the central compartment of the mitochondria and has a high protein content

  • proteins are pyruvate dehydrogenase, the enzymes of the tricarboxylic acid cycle, enzymes of fatty acid oxidation, and enzymes required for RNA, DNA, and protein synthesis

• where the mitochondrial DNA and ribosomes are located

mitochondrial genome

• mitochondria have their own genetic material; a circular DNA in higher plants and animals

• this nonchromosomal DNA encodes a small number of mitochondrial proteins that are tightly integrated into the inner membrane, along with proteins encoded by nuclear genes

• after a single aerobic bacterium took residence in the cytoplasm of a primitive cell, most of the genes of this symbiotic organism were either lost or transferred to the nucleus of the host cell, leaving only a handful of genes to encode some of the most hydrophobic proteins of the inner membrane

the function of mitochondria

• the primary function is to utilize free energy derived from oxidation of organic molecules to produce ATP under aerobic conditions

• the reaction in the mitochondria may also serve as a route to interconvert molecules of the different classes of biological molecules

  • provide intermediates

overview of carbohydrate metabolism

• coupling cytosolic glycolysis and pyruvate production to the mitochondrial TCA cycle and ATP formation

• if oxygen is present, cell will undergo glycolysis to form pyruvate, which will be transported into the mitochondria to be used to create ATP for energy

• in the absence of oxygen, the cell will undergo fermentation to produce NAD+ to be used in glycolysis

glycolysis

• the first steps in oxidative metabolism are carried out in glycolysis

• produces pyruvate, NADH, and two molecules of ATP

• aerobic organisms use O2 to extract more than 30 additional ATPs from pyruvate and NADH

• pyruvate is transported across the inner mitochondrial membrane and decarboxylated to form acetyl CoA, which enters the next stage

glycolysis pathway

know the first step, end product, and net reaction (i think)

know overall reaction but I think he means net reaction

net reaction of glycolysis

glucose + 2 NAD+ + 2 ADP +2 Pi → 2 pyruvate + 2 ATP + 2 NADH + 2 H+ + 2 H2O

the oxidative pentose phosphate pathway

• generates reducing equivalents in the form of NADPH for reductive biosynthesis reactions within cells

• provides the cell with ribose-5-phosphate (R5P) for the synthesis of the nucleotides and nucleic acids

• metabolizes dietary pentose sugars derived from the digestion of nucleic acids as well as to rearrange the carbon skeletons of dietary carbohydrates into glycolytic/gluconeogenic intermediates

importance of NADPH

• the reactions of fatty acid biosynthesis and steroid biosynthesis utilize large amounts of NADPH

• erythrocytes utilize the reactions of the PPP to generate large amounts of NADPH used in the reduction of glutathione

• the conversion of ribonucleotides to deoxyribonucleotides (through the action of ribonucleotide reductase) requires NADPH as the electron source

• therefore, any rapidly proliferating cell needs large quantities fo NADPH

pyruvate dehydrogenase complex

• the pyruvate molecules produced by glycolysis are transported across the inner membrane and into the matrix

• in the matrix they are decarboxylated by the pyruvate dehydrogenase complex to form acetyl groups, which are transferred to CoA

• complex of sixty peptides constituting three different enzymes:

  • cubelike cluster: dihydrolipoyl transacetlase molecules

  • blakc spheres: pyruvate dehydrogenase dimers

  • small gray sphere: dihydrolipoyl dehydrogenase

net reaction of pyruvate dehydrogenase

pyruvate + HS-CoA + NAD+ → Acetyl-CoA + CO2 +NADH + H+

tricarboxylic acid (TCA) cycle (aka Krebs cycle) pathway

TCA cycle

• all of the enzymes of the TCA cycle reside in the soluble phase of the matrix except for succinate dehydrogenase, which is bound to the inner membrane

• the first step in the cycle is the condensation of the two-carbon acetyl group with a four-carbon oxaloacetate to form a six-carbon citrate

• the two carbons that are removed during the TCA cycle are completely oxidized to CO2 (these two carbons are not the same ones that were brought with the acetyl group)

• in the TCA cycle, the chain length of the citrate is decreased one carbon at a time, regenerating the four-carbon oxaloacetate, which can condense with another acetyl CoA

• four reduction reactions occur in this cycle, three of which reduce NAD+ to NADH and one reduces FAD to FADH2

net reaction of the TCA cycle

acetyl CoA + 2 H2O + FAD + 3NAD+ + Pi → 2 CO2 + FADH2 + 3NADH + 3H+ + GTP + HS-CoA

catabolic pathways and the TCA cycle

• reaction intermediates in the TCA cycle are common compounds generated in other catabolic reactions

• for this reason, the TCA cycle is the central metabolic pathway of the cell

the malate-aspartate shuttle

• the primary cytoplasmic NADH electron shuttle

• mitochondria are not able to import the NADH formed in the cytosol during glycolysis

• NADH formed during glycolysis enters the mitochondria via malate-aspartate or glycerol phosphate shuttles

• these electrons are indirectly fed into the mitochondrial electron-transport chain and used for ATP formation

the glycerol phosphate shuttle

• the secondary cytoplasmic NADH electron shuttle

• electron transfer from NADH to DHAP to form glycerol 3-phosphate, then to FAD to form FADH2

  • enzyme is glycerol 3-phosphate dehydrogenase

• energy is lost; energy level of NADH is higher than that of FADH2

oxidative phosphorylation

WATCH LECTURE

• a process in which ATP formation is driven by energy that is released from electrons removed during substrate oxidation

• energy stored in the high-energy of NADH and FADH2 are released as they are passed through the electron-transport chain

substrate level phosphorylation

• a process in which ATP is formed directly by transfer of a phosphate group from a substrate molecule to ATP

oxidation and reduction

• redox couple: A/A+ and B+/B

reducing agents

oxidizing agents

oxidation-reduction potential standards

• standard conditions: concentrations are 1M for solutes and ions, and 1 atm pressure for gases

• standard couple: H+/H2

• standard redox potential: E₀

  • E₀’ - [H+] is 10^-7 (pH of 7)

• oxidizing and reducing agents occur as couples, which differ in their number of electrons

• the transfer of electrons between a couple causes charge separation that can be measured as an oxidation-reduction (redox) potential
  
  

redox potentials in the TCA cycle

• electrons are transferred to NAD+ (or FAD+) within in the mitochondrion from substrates of the TCA cycle

  • isocitrate, malate, a-ketoglutarate, and succinate

• most have redox potentials of relatively high negative values; sufficiently high to transfer electrons to NAD+

• oxidation of succinate to fumarate (more positive redox potential) proceeds by the reduction of FAD, a coenzyme of greater electron affinity than NAD+

free energy change during electron transport

sorry I didn’t feel like typing all of this

five types of electron carriers in the electron transport chain

• flavoproteins, cytochromes, copper atoms, ubiquinone, and iron-sulfur proteins

• all are membrane-bound

• all of the redox centers within the chain that accept and donate electrons are non-amino acid prosthetic groups that are tightly associated with proteins (except ubiquinone)

flavoproteins

• FMN (flavin mononucleotide) is a prosthetic group of some flavoproteins

• it is similar in structure to FAD (flavin adenine dinucleotide), but lacking the adenine nucleotide

• FMN (like FAD) can accept 2 e- + 2H+ to form FMNH2

why is FMN important

• when bound at the active site fo some enzymes, it can accept 1 e- to form the half-reduced semiquinone radical

  • the semiquinone can accept a second e- to yield FMNH2

• since it can accept/donate1 or 2 e-, FMN has an important role in mediating electron transfer between carriers that transfer 2e- (NADH) and those that can accept only 1e- (FE3+)

cytochromes

• proteins with heme prosthetic groups

• the heme iron can undergo a 1e- transition between ferric and ferrous states

  • Fe3+ + e- → Fe2+

• hemes in the three classes of cytochrome (a,b,c) differ slightly in substituents on the porphyrin ring system

three copper atoms

• all three copper atoms are located within a single protein complex fo the inner mitochondrial membrane

• the copper atoms can undergo a 1e- transition between cupric and cuprous states

  • Cu2+ + e- → Cu+

ubiquinone (UG/coenzyme Q)

• very hydrophobic

• dissolves in the hydrocarbon core of a membrane

• includes a long isoprenoid tail, with multiple units having a carbon skeleton comparable to that of isoprene

  • in human cells, usually n=10

• UQ₁₀’s isoprenoid tail is longer than the width of a bilayer

• it may be folded to yield a more compact structure, and is postulated to reside in the central domain of a membrane between the two lipid monolayers

• the quinone ring can be reduced to quinol in a 2e- reaction

  • Q + 2e- + 2H+ ←→ QH2

• when bound to special sites in respiratory complexes, CoQ can accept 1e- to form a semiquinone radical (Q-); like FMN, CoQ can mediate between 1e- and 2e- donors/acceptors

iron-sulfur proteins

• Fe-S centers are prosthetic groups containing 2, 3, 4, or 8 iron atoms complexed to elemental and cysteine S

• electron transfer proteins may contain multiple Fe-S centers

  • 4-Fe centers have a tetrahedral structure, with Fe and S atoms alternating as vertices of a cube

• Fe-S centers transfer only one electron even if they contain two or more iron atoms, because of the close proximity of the iron atoms

arrangement of electron carriers in the chain

• carriers are arranged in the order of increasingly positive redox potential

• the electrons are passed from one carrier to the next, losing energy as they move “downhill” along the chain

determination of the sequence of carriers in the chain

• the specific sequence of carriers that constitute the electron-transport chain was worked out using a variety of inhibitors that blocked electron transport at specific sites along the route

• after an inhibitor was added to cells, the oxidation state of the various electron carriers in the inhibited cells was determined

• by identifying reduced and oxidized components in the presence of different inhibitors, the sequences of the carriers could be determined

• done by Britton Chance and coworkers at the University of Pennsylvania

tendency for electron transfer

• the tendency for electrons to be transferred from one carrier to the next depends on the potential difference between two redox centers; rate of transfer depends on the catalytic activities of the proteins involved

• electrons may travel considerable distances (10-20 Å) between adjacent redox centers

  • may flow through special “tunneling pathways” consisting of a series of covalent and hydrogen bonds that stretch across parts of several amino acid residues

four complexes of the electron transport chain

• all distinct (I, II, III, IV)

• asymmetric and membrane spanning

cytochrome c and ubiquinone

• cytochrome c: a soluble protein in the intermembrane space

• ubiquinone: a pool of molecules dissolved in the lipid bilayer

• ubiquinone and cytochrome c move within or along the membrane shuttling electrons between large, relatively immobile protein complexes

• once inside the complexes, electrons travel along defined pathways between adjacent redox centers whose relative positions are fixed in space

subunits of the complexes

coupling sites

• there are three places in which transfer of electrons is accompanied by a major release of free energy; these are coupling sites

• the coupling sites occur between carriers that are part of three complexes (I, III, IV)

• the energy released at the three sites are conserved by translocation of proton from the matrix across the inner membrane into the inter-membrane space

  • these three complexes are called proton pumps

what is complex I (NADH dehydrogenase)

• the gateway to the ETC, catalyzing the transfer of a pair of electrons from NADH to ubiquinone (UQ) to form ubiquinol (UGH2)

• about half of the complex consists of a hydrophilic domain that projects into the matrix, with the remaining hydrophobic portion of the complex embedded in the membrane

• together these portions of the complex carry out the two different activities that are required of the ETC (electron transfer and proton translocation)

how does complex I work

• electron transfer occurs within the hydrophilic portion of the complex

• the passage of a pair of electrons from NADH to ubiquinone is paired with moving four protons from the matrix into the intermembrane space

• electron movement induces a conformational change in the complex that causes the lateral movement of helix HL, which leads to tilting of the transmembrane helices

• this changes the ionic environment of proton-transferring residues, leading to the movement of protons across the membrane

complex II (succinate dehydrogenase)

• consists of four polypeptides: two hydrophobic subunits that anchor the protein in the membrane and two hydrophobic subunits that comprise succinate dehydrogenase

• provides a pathway for feeding lower-energy electrons from succinate to FAD to ubiquinone

• the path from FADH2 to ubiquinone takes the electrons through three iron-sulfur clusters

• complex II contains a heme group, thought to attract escaped electrons to prevent the formation of destructive superoxide radicals

• electron transfer through complex II is not accompanied by proton translocation

complex III (cytochrome bc1)

• catalyzes the transfer of electrons from ubiquinol to cytochrome c, with four protons translocated across the membrane for every pair of electrons transferred

• protons are released into the intermembrane space in two separate steps as a pair of electrons are separated and passed along different pathways through the complex

• two protons are derived from the molecule of ubiquinol that entered the complex, and two protons are removed from the matrix and translocated as part of a second molecule of ubiquinol

• three of the subunits of complex III contain redox groups: cytochrome b contains two heme b molecules with different redox potentials, cytochrome c1, and an iron-sulfur protein

complex IV (cytochrome oxidase)

• electrons are transferred from cytochrome c through a bimetallic copper center (Cu_A) of subunit II to a heme (a) of subunit I

• electrons are passed to a redox center located in subunit I that contains a second heme (a3) and another cooper atom (Cu_B); the first two electrons reduce the a3-Cu_B) binuclear center

  • Fe_a3³⁺ + Cu_B²⁺ +2e- → Fe_a3²⁺ + Cu_B⁺

• an O2 binds to the center and accepts the pair of electrons

  • Fe³⁺-O^- - O^- - Cu²⁺

• a third electron was extracted and the binuclear center accepts two protons

  • one of the O is reduced: Fe⁴⁺ = O^2- Cu²⁺ -OH2

• a fourth electon and two additional protons are used to form two molecules of water

  • Fe³⁺ Cu²⁺ + 2H2O

• four additional protons are pumped across the inner membrane to the intermembrane space

experiment showing that cytochrome oxidase is a proton pump

• cytochrome oxidase: a proton pump in synthetic liposomes

• the medium becomes acidified following the addition of reduced cytochrome C

• translocation of protons across the inner membrane produces electrochemical gradient:

  • the concentration difference of hydrogen ions (deltapH)

  • the voltage resulted from the separation of charge (ψ)

• the proton-motive force (deltap):

  • deltap = ψ -2.3(RT/F) deltapH

  • where 2.3 TR/F is equal to 59 mV at 25 degrees C:

    • deltap = ψ =59 deltapH

• positively charged, lipid-soluble substance (JC-1) is distributed according to the electric potential; the voltage genderated across the inner membrane (inside negative) leads to the accumulation of the dye within the mitochondria

ADD PICTURES!!

F1 and ATPase

• during the early 60s, using negative staining technique, Humberto Ferdandez-Moran discovered a layer of spheres attached to the inner side of the inner membrane, projecting from the membrane and attached to it by stalks

• a few years later, Efraim Rocker isolated the inner membrane spheres (coupling factor 1 or F1)

• he found that the F1 behaved like an ATPase

• F1 could be the ATPase because:

  • enzymes do not affect the equilibrium constant of the reaction they catalyze

  • enzymes are capable of catalyzing both the forward and reverse reactions

Na+/K+ ATPase

• under physiological conditions, Na+/K+ ATPase utilizes the energy from ATP hydrolysis to export Na+ and import K+ against their respective gradients

• red blood cells ghosts are prepared with a very high internal [K+] and a very high external [Na+]

• under these conditions, K+ moves out of the cell and Na+ moves into the cell

• in the presence of ADP and Pi, the movement of the ions can cause ATP to be synthesized

structure of ATP Synthase

• a spherical F₁ head (about 90 Å in diameter)

• a basal section, F₀, embedded in the inner membrane

• a central and spherical stalk connecting F₁ and F₀

F₁ structure

• has a stoichiometry of α₃β₃δγε

• the α and β subunits are arranged alternately within the F₁ (looks like pieces of an orange)

• each of the β subunits contain a catalytic site for ATP synthesis

• the γ subunit runs from the outer tip of the F₁ head through the central stalk and makes contact with the F₀ base piece

F₀ structure

• has a stoichiometry of ab₂c_(10-14)

• the number of subunits in the c ring varies from 10-14

• both the yeast and e. coli ATP synthase have 10 c subunits, whereas the chloroplast one has 14

• the F₀ base contains a channel through which protons are conducted from the intermembrane space to the matrix

evidence that the F₀ base contains a channel through which protons are conducted

• intact submitochondrial particles, which contain the ATP synthase embedded in the vesicle membrane, are capable of oxidizing substrates, generating a proton gradient, and synthesizing ATP

• if the F₁ spheres are removed from the particles, the vesicle membrane can no longer maintain a proton gradient despite continuing substrate oxidation and electron transport

Paul Boyer

• proposed a hypothesis for how a proton electrochemical gradient provides the energy required to drive the synthesis of ATP

• called the binding change mechanism

the binding change mechanism

1. the energy released by the movement of a proton is not used to drive ADP phosphorylation directly but principally to change the binding affinity of the active site for the ATP product

• enzyme-bound ADP and enzyme-bound Pi readily condense to form a tightly bound ATP molecule without the input of additional energy; instead, energy is required for the release of the tightly bound ATP from the catalytic site

2. each active site progresses successively through three distinct conformations that have different affinities for substrates and products

• the F₁ complex has three cat. sites

• at any given instant, one site is in the “loose” (L conf.) in which ADP and Pi are loosely bound; a second site is in the “tight” (T conf.) in which nucleotides (ADP+Pi or ATP) are tightly bound; and the third site in the “open” (O conf.) which allows release of ATP

3. ATP is synthesized by rotational catalysis in which one part of the ATP synthase rotates relative to another parts

• α and β subunits rotate relative to the central stalk; the rotation is driven by the movement of protons through the membrane via the channel in the F₀ base

binding change mechanism photo

evidence to support the binding change mechanism

• in 1994 John Walk and his co-workers published a detailed atomic model of the F₁ head

• structures corresponding to the L, T, and O conformations were identified in the cat. sites of the 3 β subunits

• the γ subunit is perfectly positioned within the ATP synthase to transmit conformational changes from F₀ membrane sector to the F₁ cat. sites; the γ subunit extends from the F₀ sector through the stalk and into the central cavity within the F₁ sphere, where it contacts each of the three β subunits differently

• in the presence of ATP, the actin filament was observed to rotate counterclockwise

• the β subunit was modified to contain 10 histidine residues at its N-terminus, as site located on the matrix face of the F₁ head

• the γ subunit was modified by replacing one of the serine residues near the end of the stalk with a cysteine residue, which provided a means to attach the fluorescently labeled actin filament

the structure and function of the membrane-bound F₀ portion

• what is the path taken by protons as they move through the F₀ complex and how does this movement lead to the synthesis of ATP? it was postulated that:

1. the c subunits of the F₀ base were assembled into a ring that resides within the lipid bilayer

2. the c ring is physically bound to the γ subunit of the stalk

3. the “downhill” movement of protons through the membrane drives the rotation of the ring of c subunits, and the rotation of the c ring of F₀ provides the twisting force that drives the rotation of the attached γ subunit, leading to the synthesis and release of ATP

• the β subunits are primarily structural components of the ATP synthase

• the two elongated β subunits form a peripheral stalk that connects the F₁ and F₀ portions, and along with the δ subunit of F₁, hold the α₃β₃ subunits in a fixed position while the γ subunit rotates within the center of the complex

• each c subunit contains two transmembrane helices connected by a hydrophilic loop that projects toward the F₁ head; the hydrophilic loops situated on the tops of the c subunits form a binding site for the bases of the γ and the ε subunits, which together act like a “foot” that is attached to the c ring

  • as a result of this attachment, rotation of the c ring drives rotation of the attached γ

subunit a of the F₀ portion

• subunit a is stationary; it has two half-channels that are physically separated from one another

• one half-channel leads from the intermembrane space into the middle of the a subunit and the other leads from the middle of the a subunit into the matrix

• the subunits of the c ring move successively past the stationary a subunit

• protons are picked up from the intermembrane space one at a time by each c subunit and carried completely around a circle before they are released into the matrix

• each proton moves from the intermembrane space through the half-channel and binds to a negatively charged Asp residue situated at the surface of the c subunit

• at the same time, the adjoining c subunit (which was protonated at an earlier step) is aligned with the second half-channel of the a subunit

• the Asp releases its associated proton, which diffused into the matrix

• after dissociation of the proton, the c subunit returns to its original conformation

• binding of the proton to the carboxy group generates a major conformational change in the c subunit that causes that subunit to rotate approx. 30 degrees in a counterclockwise direction

• the movement brings the adjoining c subunit, which has just released the proton, into alignment with the other half-channel and repeat the cycle

how many molecules of ATP comes from ATP synthase

• if a c ring is composed of 12 subunits, it will require 4 protons to move the ring 120 degrees, which would drive a corresponding rotation of the γ subunit 120 degrees and lead to the release of one newly synthesized ATP by the F₁ complex

• therefore, the translocation of 12 protons would lead to the fill 360 rotation of the c ring and γ subunit, and the synthesis and release of 3 molecules of ATP

other processes driven by proton-motive force

• uptake of ADP/Pi in exchange for ATP and H+; ATP/ADP exchange is accomplished by the adenine nucleotide translocase (ANT)

• pull Ca2+ ions into the mitochondrion

• drive mitochondrial fusion

• specifically targeting polypeptides to enter the mitochondrion from the matrix

• pumping pyruvate into the matrix (symport)

photo shows a summary of the major activities during aerobic respiration in a mitochondrion

peroxisomes

• membrane-bound vesicles that contain oxidative enzymes

• the oxidize very long chain fatty acids, and synthesize plasmalogens (a class of phospholipids)

• they form by splitting from pre-existing organelles, import preformed proteins, and engage in oxidative metabolism

• hydrogen peroxide, and reactive and toxic compound, is formed in peroxisomes and is broken down by the enzyme catalase

  • peroxisomes contain enzymes to carry out the two-step reduction of molecular oxygen to water

glyoxysome

• a specialized type of peroxisome in plant seedlings

• plant seedlings rely on stored fatty acids to provide the energy and material to form a new plant

• one of the primary metabolic activities in these germinating seedlings is the conversation of stored fatty acids to carbohydrate

• citrate is converted into glucose by a series of enzymes of the glyoxylate cycle localized in the glyoxysome