Explain how the orientation of an amino acid in the binding site of a PLP-containing enzyme determines the stereoelectronic control of the reaction that will take place at the ?-carbon of the amino acid (e.g., transamination, decarboxylation, racemization
It all depends on the position in the active site of the enzyme. The bond that is broken depends on position. The bond most nearly perpendicular to the p-orbitals is the one that is broken. Whatever bond on the alpha carbon is the most perpendicular to th
Explain the reactions (specific substrates, specific products, specific enzyme names, specific cofactors, specific reaction types) of the citric acid cycle; you should be able to draw and recognize these substrates & products, as well as where ATP/GTP, NA
See wall as well.
The acetyl CoA comes from the PDH and a pyruvate from glycolysis.
Pneumonic: Citrate Is A Super Substrate For Making Oxaloacetate.
1.) Citrate synthase
2.) Aconitase
3.) Isocitrate dehydrogenase (NADH + H+)
4.) Alpha-ketoglutarate dehydr
Describe the purpose of each individual reaction of the citric acid cycle; that is, the chemical logic and purpose of each step
1.) C-C bond formation to make citrate
2.) Isomerization via dehydration/rehydration
3-4.) Oxidative decarboxylations to yield 2 NADH
5.) Substrate-level phosphorylation to yield ATP (or GTP). GTP is liver and ATP is muscle. No longer distinguish carbons
Summarize the specific intracellular location of each enzyme of the TCA cycle
Everything occurs in the mitochondrial matrix EXCEPT the Succinate dehydrogenase reaction, which occurs on the inner membrane of the mitochondria.
Identify the metabolically irreversible reactions of the TCA cycle & which reactions are close to equilibrium
The first (citrate synthase) and fourth step (alpha-ketoglutarate dehydrogenase complex) and Isocitrate dehydrogenase are highly irreversible and spot for regulation.
Step 2, 5, 6, and 7 are close to equilibrium.
Track individual carbons through the citric acid cycle until asymmetry is lost (should be to do starting from any glycolytic intermediate)
Just know based of previous knowledge. The CO2 that is lost from pyruvate is derived from the 4 or 3 carbon of glucose. The carbons on acetyl CoA are from 6, 1, 5, or 2.
Both CO2 carbon atoms are derived from oxaloacetate. The acetyl-CoA carbons stay unti
Recognize induced fit of substrates in citrate synthase
CITRATE SYNTHASE IS THE RATE LIMITING STEP OF CITRIC ACID CYCLE. Activity depends on [Oxaloacetate].
The conformational change occurs upon binding of oxaloacetate and it avoids unecessary hydrolysis of thioester in acetyl-CoA.
The binding of oxaloacetate
Recognize that aconitase has a Fe-S center in its active site
Aconitase catalyzes a reaction that sets up a Beta-bond (Isocitrate) to the carbonyl so no cofactor is required. Addition of H2O to cis-aconitase is stereospecific.
Water removal and addition are catalyzed by Fe-S center, which is sensitive to oxidative s
Explain the two oxidative decarboxylations in the TCA cycle and compare them
The two decarboxylation steps are the 3 and 4 step (Isocitrate dehydrogenase and alpha-ketoglutarate dehydrogenase complex).
Isocitrate dehydrogenase reaction is CRITICAL for determining OVERALL RATE of TCA cycle. The oxidative decarboxylate leads to CO2
Compare ?-ketoglutarate dehydrogenase complex to pyruvate dehydrogenase complex with respect to cofactors, mechanism, etc.
Everything is the same as the PDC (cofactors, order, everything). The active sites are the only things that are different due to the different sizes of the substrates.
Explain what is mean by substrate-level phosphorylation (this is in stark contrast to what we will see in oxidative phosphorylation via ATP synthase)
If GTP is created, the the phosphoryl group is transferred to ADP to create ATP via a nucleoside diphosphokinase.
Succinyl-CoA synthetase is an example of a thermodynamically coupled reaction to form GTP (or ATP).
Energy of thioester allows for incorporat
Interpret an enzyme's function from its name
Synthase: Catalyzes condensation reaction in which no NTP is required as an energy source. Class of ligase.
Synthetase: Catalyze condensation reactions that DO use NTP as an energy source. Form of ligase.
Ligase: Catalyze condensation reactions in which t
Describe the steps of the methylene oxidation sequence (& compare this to ?-oxidation of fatty acids [Chap 22 which is coming up])
Create a methylene species (Fumarate) and then oxidize it from malate to oxaloacetate. Use FAD.
Asymmetry is lost with succinate, can no longer distinguish carbons.
Summarize the net result of the TCA cycle
Only operates under aerobic conditions.
2 CO2, 3 NADH, FADH2, ATP (or GTP in liver), CoA, and 3 H+. Equivalent to two carbons of acetyl-CoA BUT NOT the exact same carbons.
Describe a metabolon & substrate channeling and why these would be advantageous
A metabolon is a highly organized, multi-enzyme complex that facilitates substrate channeling. The cellular environment is very crowded and you need to have these complexes to help speed up reactions and make sure they are used by the right enzymes. Diffu
Describe the allosteric & covalent regulation of the TCA cycle: specific enzymes, positive & negative effectors, the kinases & phosphatases, and their substrates
Regulation occurs at the Pyruvate DH, isocitrate DH, and alpa-ketoglutarate DH since these are highly thermodynamically favorable.
Activated by substrate availability and inhibited by product accumulation. Inhibitors are NADH and ATP, Activators are NAD+
Compare/contrast this regulation with respect to tissue and state (e.g., liver vs active muscle vs resting muscle)
Muscle at rest: The high energy charge promotes phosphorylation of PDH which will INACTIVATE it.
Exercising muscle: Low energy charge and will promote DEphosphorylation of PDH which will ACTIVATE it.
If isocitrate DH is inhibited, citrate can go to cytopl
Summarize what is meant by amphibolic pathways and present a few examples of amphibolic intermediates/reactions
It describes a biochemical pathway that involves both catabolism and anabolism. Krebs cycle and PPP are examples.
Summarize what is meant by anaplerotic reactions and why they are critical for maintaining metabolic homeostasis; provide four examples of anaplerotic reactions
Chemical reactions that form intermediates of a metabolic pathway.
The intermediates produced in TCA cycle can be used in other biosynthetic pathway, but they must be replenished for the cycle and central metabolic pathway to continue to maintain homeosta
Interpret the roles of TCA intermediates under the following conditions: when energy is required, when energy needs are satisfied, when oxaloacetate synthesis is required (how & why)
When cell needs energy: The CAC will operate as normal and will go on to the electron transport chain.
When cell energy needs are met: The intermediates are used for biosynthesis (Fatty acids, sterols, amino acids, heme, porphyrins, glucose, purines, pyri
Identify, know, & draw the structure of palmitate
16:0 CH3(CH2)14COO-
Compare & contrast fatty acids & carbohydrates as fuel sources with respect to initial oxidation state, hydration, long-vs short-term need & use, intracellular location & tissue type of storage (glycogen vs TAGs), ATP yield, etc.
Roughly 1/3 of our energy needs comes from dietary TAGs
80% of energy needs needs of mammalian hearts and liver are met by oxidation of fatty acids
Hibernating animals rely heavily on fats
Fatty acids carry more energy per carbon because they are highly r
Describe triacylglycerol structure & chemical linkages
They have ester linkages and it has a glycerol backbone. Three fatty acids connected to the backbone.
Describe how TAGs are converted to DAGs which are converted to MAGs; that is, through hydrolysis via lipases with release of fatty acids
TAG is converted to DAG via a pancreatic lipase and a hydrolysis reaction that gives off a free fatty acid. The DAG is then converted to MAG via another pancreatic lipase hydrolysis reaction and the release of another fatty acid.
Summarize the digestion of dietary fatty acids: bile salts (purpose, where they are derived from & where synthesized); role of pancreatic lipases in intestinal lumen (what are substrates & products, & purpose of these reactions); role of mucosal cell in p
First fatty acids enter the small intestine where bile salts emulsify them and form mixed micelles. Bile salts are synthesized from cholesterol in liver and secreted by the gallbladder. They solubilize dietary fats to increase the area exposed to lipases
Summarize the mobilization of TAGs in response to hormone (e.g., glucagon or epinephrine) and regulation of the following: perilipin, adipose triglyceride lipase, HS-lipase
Hydrolysis of TAGs is catalyzed by lipases in adipocytes. Some lipases are regulated by hormones glucagon and epi. Epi means energy NOW, whereas Glucagon means OUT of GLUCOSE.
Perilipin is a fat-droplet associated protein and phosphorylation from PKA rest
Summarize lipolysis: where glycerol & fatty acids go & their fates
Lipolysis generates Glycerol and Fatty acids. Glycerol goes on to hepatocytes where it can enter Glycolysis or gluconeogenesis via glycerol kinase which then converts into DHAP and then GAP. Fatty acids go to other tissues and go through beta-oxidation an
Explain the role of lipases in TAG mobilization and how this mobilization facilitates transport of fatty acids (not TAGs) across the adipocyte plasma membrane & into blood
Lipases degrade TAGs into Fatty acids and MAG (glycerol) for transport into the cell since TAGs cannot cross the plasma membrane freely.
Summarize the role of serum albumin in fatty acid transport
Serum Albumin binds to fatty acids for transport in the blood.
Explain how fatty acids cross the plasma membrane of the target cell (e.g., myocytes): fatty acid transporter
TAGs are degraded into fatty acids and glycerol in CYTOPLASM of adipocytes. The fatty acids are then transported via serum albumin to other tissues and they enter the tissues via fatty acid transporters in the plasma membrane.
Describe the fate of glycerol (tissue location) and reactions & how the subsequent products feed into glycolysis/gluconeogenesis) post-TAG mobilization
Glycerol goes into the hepatocytes where glycerol is first activated into glycerol-3-phosphate via glycerol kinase. This L-glycerol-3-phosphate is oxidized into DHAP via Glycerol Phosphate DH. DHAP then converts to GAP, and then it enters either glycolysi
Summarize the reaction (& intracellular location) of fatty acid's conversion to its CoA derivative (specific enzyme name, cofactors, intermediate [acyl adenylate], reactants & products) and how the coupled PPi hydrolysis drives the overall reaction
Occurs in the cytosol of cells. The fatty acid attacks the alpha phosphate of ATP to form an Acyl Adenylate intermediate and a pyrophosphate. The PPi is immediately hydrolyzed to two molecules of Pi. The fatty acyl-adenylate (enzyme bound) intermediates i
fatty acyl adenylate intermediate
It is a mixed anhydride and enzyme bound.
Compare how shorter & longer chain fatty acids enter the mitochondrial matrix
Small (<12 carbons) fatty acids diffuse freely across the mitochondrial membranes.
Larger fatty acids (most free fatty acids) are transported via acyl-carnitine/carnitine transporter.
Explain the role of carnitine, intracellular locations & function of acyl-carnitine transporters
Carnitine shuttles long fatty acids into the mitochondrial matrix. It is like the cloak for long fatty acids to get in to the MM.
First, carnitine is membrane bound and then by Carnitine acyltransferase 1, the Fatty acid from Fatty acyl-CoA is transferred
Describe the steps of ?-oxidation of fatty acids (& compare and make analogies to the specific enzymes catalyzing the methylene oxidation sequence in the TCA cycle): intracellular location; CoA activation, methylene oxidation (general enzyme names only) w
Each pass removes one acetyl moiety in the form of acetyl-CoA.
1.) Dehydrogenase using FAD catalyzes the dehydration of alkane to alkene. Enzyme on inner mitochondrial membrane. Results in trans double bond and is analogous to succinate DH reaction in CAC
Summarize with respect to acyl CoA dehydrogenase: acyl chain length specificity; trans double bond formation (why this); cofactor being reduced
This enzyme can do long-chains (12-18), medium chains (4-14) and short chains (4-8) [Do not need to know exact lengths]. The product is a trans double bond and it is different from naturally occurring unsaturated fatty acids. FAD is being reduced to FADH2
Describe how electrons are transferred from acyl CoA dehydrogenase to ubiquinol (that is, if you were given a sequence of electron transport coupled reactions, you should be able to put the arrows in the correct direction for electron transfer)
Goes from acyl-CoA to Ubiquinol, which then delivers electrons to oxidative phosphorylation. See wall for the chain but just base arrows off what is reduced and what is oxidized. Not too hard. Something being oxidized, something has to be reduced.
Analyze the number of FADH2 & NADH being produced and the number of CoAs required if given a fatty acid of a specific length or saturation (e.g., 12:0 fatty acid)
Whatever length the chain is (if saturated), just divide by 2 and that will give you the amount of Acetyl-CoA produced. The number of cycles is always ONE LESS than the amount of Acetyl-CoA produced since at the last cleavage, 2 Acetyl-CoA's are produced.
Compare & contrast the degradation of monounsaturated & polyunsaturated fatty acids (what general enzyme names used for what type of reaction) with saturated fatty acid degradation
The unsaturated fatty acids need two additional enzymes, an isomerase that converts cis double bonds at C-3 to trans double bonds, and a reductase that reduces cis double bonds not at carbon 3 (Uses NADPH).
MONOunsaturated only need the ISOMERASE
POLYunsa
Compare & contrast even & odd numbered fatty acid degradation
Even you get nice number of acetyl-CoA. Many plants and some marine organisms synthesize odd numbered fatty acids. At the end, you are left with an Acetyl-CoA and a Propionyl-CoA (3 carbon). The propionyl-CoA then needs to be further oxidized to produce S
Explain propionyl CoA oxidation: propionyl CoA carboxylase (know specific enzyme name, cofactors required, make analogies to pyruvate carboxylase); epimerase (general name); methyl-malonyl CoA mutase (know specific enzyme name, cofactor & cobalamin + Co3+
Propionyl-CoA (3C) has to be converted to Succinyl-CoA (4C) to enter the CAC.
1.) Propionyl-CoA is converted to D-Methylmalonyl-CoA via carbonate (HCO3-) and ATP and Biotin cofactor. ADP and Pi are created in the process. Like Pyruvate carboxylase reactio
Describe the reactions catalyzed by mutases
Mutases catalyze a group transfer reaction. One group moves from one position to another. Example is 3-Phosphogluconate to 2-Phosphogluconate.
Compare & contrast ?-oxidation of fatty acids in mitochondria & peroxisomes: fate of electrons from FADH2 & NADH, fate of acetyl CoA
Mitochondria: The acyl-CoA dehydrogenase passes electrons into the respiratory chain via electron-transferring flavoprotein (ETF) where energy is created in the from of ATP.
Peroxisomal/Glyoxysomal: The acyl-CoA DH passes electrons directly to O2 and ener
Summarize conversion of acetyl CoA to ketone bodies: relationship to oxaloacetate; physiological conditions when required & their physiological purpose; tissue & intracellular location of synthesis & where exported to; the three specific molecules that ar
If carbohydrates are unavailable or improperly used, ketone bodies will be formed. Ketone bodies are fuel source for various tissues during starvation or untreated diabetes. Occurs in hepatocytes. Acetoacetate, Acetone, and D-?-hydroxybutyrate are the thr
Explain activation of acetyl CoA via acetyl CoA carboxylase: intracellular location; purpose of this reaction; compare to pyruvate carboxylase & propionyl CoA carboxylase reactions; activation of biotin by ATP & CO2 binding; role of tethering
Reaction takes place in the CYTOSOL. It carboxylates acetyl-CoA via Acetyl-CoA carboxylase. Bicarb is the source of CO2. The CO2 binds to biotin after activation by ATP to produce a carbamoyl phosphate. The CO2 is first added to the biotin, then the arm s
Know & draw the structure of malonyl CoA
When synthesizing fatty acids, one acetate unit is processed at a time and the acetate is coming from activated malonate in the form of malonyl-CoA.
Describe the steps of fatty acid synthesis: intracellular location; acyl carrier protein activation, condensation, reduction of keto to methylene; NADPH as cofactor; names of enzyme activities of fatty acid synthase; the roles and locations of the two thi
Occurs in cytosol, but have to transport Acetyl-CoA out of MM. Catalyzes a repeating four step sequence that elongates the fatty acyl chain by two carbons at each step. Uses NADPH as electron donor. Uses two enzyme bound -SH groups for activation. Only FA
Explain the four reactions of FAS and activation with ACP
Prep: ACP and FAS must be charged with Acyl group. Thiol from 4-phosphopantethin in ACP and thiol from Cys in FAS are spots for charging. Acetyl group of Acetyl-CoA is transferred to ACP by MAT. ACP then passess this acetate to the Cys of the ?-keto synth
Explain the role of ACP and its tethering
It is a Macro-CoA. Contains a 4'-phosphopantetheine prosthetic group which is the flexible arm that swings intermediates from one active site to another. Delivers acetate (first step) or malonate (all other steps) to the FAS. Shuttles growing chain from o
Analyze the number of acetyl CoAs & ATPs consumed and NADPH required by fatty acid synthase if given a fatty acid of a specific length to synthesize (e.g., 12:0 fatty acid) and ALSO BE SURE
to add in ATP cost of acetyl CoA transport out of mito into cytos
For palmitate (16:0), Seven cycles produces this fatty acid. In the process, 7 acetyl-CoAs are used to create 7 malonyl-CoAs at the expense of 7 ATP. Then, 1 Acetyl-CoA is used, along with 7 malonyl-CoA and 14 NADPH, to create palmitate. Have to use 1 mor
fatty acyl-CoA desaturase
Occurs in cytoplasmic side of ER membrane. It is catalyzed by Fatty acyl-CoA desaturase and it can only from a double bond between carbons 9 and 10. It requires NADH, cytochrome b5, and cytochrome b5 reductase. Mammals cannot put double bonds past Carbon
Compare & contrast NADPH sources (pentose phosphate pathway and malic enzyme) with respect to tissue & intracellular location
NADPH levels are high in cytosol so that is why fatty acid synthesis takes place there. Chloroplast are high in NADPH in plants.
Adipocytes: PPP and Malic enzyme create NADPH.
Hepatocytes and mammary gland: PPP.
Plants: Photosynthesis.
Explain the specific reaction catalyzed by malic enzyme (substrates, products, cofactors)
Malate is converted to pyruvate and CO2. This creates NADPH and an H+. The decarboxylation drives the reaction forward.
Describe how acetyl CoA is shuttled from the mito matrix into the cytosol: specific enzyme name, substrates, products & cofactors, citrate transporter location; specific conversion of citrate into acetyl CoA & oxaloacete via citrate lysase & its ATP requi
Acetyl-CoA is converted to citrate. The first reaction in the CAC converts acetyl-CoA and oxaloacetate into citrate. Catalyzed by citrate synthase. The citrate can then leave the MM via the citrate transporter and then enter the cytosol. The Citrate that
Summarize oxaloacetate's conversion to malate
Oxaloacetate in the cytosol is then converted to malate. Malate dehydrogenase reduces oxaloacetate to malate. Oxaloacetate could go on to gluconeogenesis as well.
Compare & contrast malate's subsequent fates: via malic enzyme & via malate/?-ketoglutarate transporter
1.) Can be converted to NADPH and pyruvate via malic enzyme. The NADPH is used in lipid synthesis and the pyruvate can be sent back to MM via pyruvate transporter and converted back to oxaloacetate by pyruvate carboxylase.
2.) Can be transported back into
Integrate pentose phosphate pathway, fatty acid synthesis & degradation, glycolysis with respect to intracellular location and key intermediate & cofactor relationships
Glycolysis, fatty acid synthesis/degradation and PPP occur in the cytosol. PPP creates NADPH for synthesis of fatty acids. Malate can create NADPH for synthesis of fatty acids via malic enzyme. Know other reactions to compare and contrast.
Describe the allosteric & covalent regulation of acetyl CoA carboxylase in fatty acid synthesis: positive & negative effectors, the kinases & phosphatases and their substrates
Acetyl-CoA carboxylase catalyzes the rate-limiting step. It is inhibited by high levels of palmitoyl-CoA and activated by citrate. Citrate signals excess energy that can be converted to fat when [Acetyl-CoA] in mitochondria is high. Citrate then exported
Explain the role of citrate in regulating ACC and PFK-1
When Acetyl-CoA and ATP are high, citrate is sent to cytosol to be cleaved into acetyl-CoA and do fatty acid synthesis.
It is an allosteric activator of acetyl-CoA carboxylase (ACC)
It is an inhibitor of PFK-1 (Reduces glycolysis).
Compare and contrast the effects of insulin & glucagon on fatty acid synthesis and degradation
Glucagon: Reduce sensitivity of citrate acitivation and leads to phosphorylation (Inactivation) of ACC. Fatty acids are broken down and glucose is released to body.
Insulin: Activates the phosphatase and it dephosphorylates ACC to make it active. Will the
Explain the inhibitory effect of malonyl CoA on the acyl-carnitine
transerase I (so no enter the mito!)
If there is an excess of Malonyl-CoA, it will inhibit Carnitine Acyl-Transferase I so that the oxidation of fatty acids does not occur and they cannot go into the MM. Excess means you are trying to synthesize fatty acids, not break them down.
Describe what is meant by essential amino acids (do NOT need to memorize which ones are essential and which are non-essential aa's)
90% of energy needs of carnivores can be met by amino acids immediately after a meal.
Dietary amino acids that exceed body's proteins synthesis needs display need for oxidation.
Proteins in the body can be broken down to supply AA's for energy when carbs
Summarize the digestion of dietary protein: pepsin & location; trypsin/chymotrypsin & location; aminopeptidases & carboxypeptidases & location
Pepsin cuts proteins into peptides in the stomach. Chief cells in stomach lining secrete pepsinogen (inactive form of pepsin).
Trypsin/Chymotrypsin cut proteins and larger peptides into smaller peptides in the small intestine. Secreted by the pancreas. Se
Describe the pros & cons of protein degradation
Half lives of proteins vary a lot. Hemoglobin is long lived and defective or metabolism regulatory proteins are short-lived.
Good: Activate or turn off a signaling pathway which eliminates damaged proteins.
Bad: Protein aggregation can lead to diseases su
Summarize the ubiquitin-degradation pathway: the three specific enzymes (names & roles) that add ubiquitin; the role of ubiquitin; the roles of the 20S & 19S units of the 26S intact proteasome; the outputs after proteasome digestion & subsequent fates of
Proteins are linked to Ubiquitin via ubiquitin-activating enzyme (E1, adenylation reaction), Ubiquitin-conjugating enzyme (E2, transfer to a Cys on E2), and Ubiquitin-protein ligase (E3, Transfer to Lys on target). Ubiquitinated proteins are then cleaved
Describe the shape of the 26S proteasome
The 20S has two alpha subunit caps on either end, and the beta subunits in the middle. The beta subunits is where the business happens and some have protease sites.
Compare a peptide bond with an isopeptide bond
An isopeptide bond is an amide bond that can form from side chain amino acid residues. They are not part of an alpha group.
A peptide bond is an amide bond where the alpha groups (Carboxyl and Amine) are covalently bonded.
Explain what a degron is & its role in protein degradation
Degron: A specific amino acid sequence that regulates protein degradation rates. There is the N-terminal rule or pro-N-terminal degron. E3 READS these degrons.
Cyclin Destruction Boxes: Mark cell-cycle proteins.
PEST sequences can be degrons (PEST are sin
Describe the first step of amino acid degradation: removing nitrogen via transamination of ?-ketoglutarate (using an aminotransferase [as known as, transaminase])
Removal of the amino group is the first step in degradation for all AA's.
Use a Pyridoxal Phosphate (PLP) cofactor. Typically, alpha-ketoglutarate is the acceptor of amino groups.
L-glutamate acts as a temporary storage for N, and L-glutamine can donate a
Know aminotransferases use PLP as a cofactor in a very different manner than we saw with phosphorylase's use of PLP
Instead of transferring carbon units, it transfers an amino group. Still attached to enzyme via a Schiff base at a Lys residue (Imine functional group).
Summarize the aldehyde form of PLP reacting with amino groups & the aminated form of PLP reacting with carbonyl groups
The aldehyde form can react reversibly with amino groups. The aminated form of PLP (Pyridoxamine phosphate) can react reversibly with carbonyl groups; it is an intermediate.
Compare the internal aldimine of PLP with the external aldimine of PLP
The internal aldimine is how the PLP is covalently linked to the enzyme in the resting state. Linkage is made via a nucleophilic attack of the amino group of an active site Lys. Amino acids displace the internal aldimine. The aldimine becomes external whe
Identify a PLP-Schiff base and explain why it is a good electron
sink
PLP-amino acid Schiff base is a good electron sink due to the possibilty of resonance and rearrangement possibilities. Lots of arrows can be drawn with movement of electrons.
Describe the general transamination reaction that is facilitated by PLP
The internal aldimine is formed by a Lys residue on the enzyme with PLP. The amino acid then comes in and displaces the Lys residue, and forms a protonated Schiff base with PLP, forming an external aldimine.
Summarize oxidative deamination via glutamate dehydrogenase (know specific enzyme name): tissue & intracellular location; cofactors; substrate & products; fate of ammonia
Occurs within the mitochondrial matrix of LIVER. Can use either NAD+ or NADP+ as electron acceptor. Ammonia is processed into Urea for excretion. Glutamate DH catalyzes the transformation back to alpha-ketoglutarate and the NH4+ is off to the urea cycle.
Define ureotelic organism; that is, in what form do these organisms excrete nitrogen?
Excrete Nitrogen as Urea. Many terrestrial vertebrates and sharks. Far less toxic than ammonia and highly soluble.
FYI: Humans and great apes excrete both urea (from AA's) and Uric acid (Purines).
Identify & draw the structure of urea
Excess water.
Identify & draw the structure of carbamoyl phosphate
Recall that muscle degrades branched amino acids as fuel under vigorous exercise
Muscles catabolize branches AA's during fasting and prolonged exercise. Amino acids broken down in muscle are transported in the blood as glutamine and alanine. Leucine, Isoleucine, and valine are oxidized in the muscles, adipose, kidney, and brain.
Pyruv
Summarize paths of how nitrogen is transported to liver for processing via the urea cycle: transport via glutamine (glutamine synthesis in muscle; glutaminase in liver mitochondria); transport via alanine
Two paths for Nitrogen to get to liver from muscle.
1.) Glucose-alanine cycle
2.) Transport using Glutamine (Gln). Glutamine synthetase in the muscle which then is processed by glutaminase in the liver mito.
glucose-alanine cycle
Glycolysis: Breaks down glucose into pyruvate in CYTOSOL
Amino acid Degradation: Transamination occurs in CYTOSOL (Muscle and liver) and oxidative deamination occurs in MITOCHONDRIAL MATRIX of hepatocytes.
Gluconeogenesis: Occurs in part MM at beginning a
Explain that alanine transfers its N to ?-ketoglutarate in liver cytoplasm; glutamate is transported into mito matrix and is deaminated via glutamate dehydrogenase (see above)
See slides and wall for detail. See picture. Glutamate forms an NH4+ that goes on to the urea cycle and then an alpha-ketoglutarate from the glutamate DH reaction.
Recognize that carbamoyl phosphate is synthesized in hepatocyte mitochondrial matrix from NH4+ (resulting from reaction catalyzed by glutamate dehydrogenase) & CO2 via carbamoyl phosphate synthetase & consumes 2 ATPs
Carbamoyl phosphate synthetase captures free ammonia in the MM and is the first step of the urea cycle. Highly regulated. The ammonia is from the glutamate. Carbamate intermediate gets phosphorylated to carbamoyl phosphate (mixed anhydride).
Summarize urea cycle: know what reaction occurs in mito matrix & which reactions in cytoplasm; know order (KNOW NAMES of blue molecules, BUT NOT structures of the blue molecules) of urea cycle (see Berg Fig 23.16 in notes and text); know how Asp & fumarat
See wall also and notes. Names to know in order for cycle: Ornithine goes to citrulline (MM), Citrulline goes to Argininosuccinate (Cytosol), Argininosuccinate goes to arginine (Cytosol), Arginine goes to ornithine (Cytosol). Know and draw Fumarate and As
Know conditions required to upregulate the expression of urea cycle enzymes
Carbamoyl synthetase is activated by N-acetylglutamate. This is formed by when [Glutamate] and [Acetyl-CoA] are high; activated by arginine.
Expression of enzymes increases when they are needed. For example, a high protein diet or starvation.
Know oxaloacetate is transaminated to Asp in mito matrix & transported to cytoplasm; Asp donates the second N to eventually form urea via argininosuccinate
Aspartate aminotransferase converts oxaloacetate into aspartate. Glutamate is the amino donor and it will form alpha-ketoglutarate. Aspartate in the mitochondrial matrix is then transported to the cytosol where it reacts with the Citrullyl-AMP intermediat
Know what the molecular donors are for the two N's and the C in urea
One N comes from NH4+, One N comes from Asp, and the C comes from bicarbonate (HCO3-) in the carbamoyl phosphate reaction.
Compare & contrast what is meant by ketogenic & glucogenic
amino acids
Ketogenic amino acids can be converted to ketone bodies (Acetyl-CoA, fatty acid stuff).
Glucogenic amino acids can be converted to glucose (Pyruvate, alpha-ketoglutarate, succinyl-CoA, Fumarate, oxaloacetate).
Describe the following specific transformations & interpret the fates of these specific carbon skeletons (glucogenic or ketogenic): Asp ? fumarate (viaargininosuccinate); Asp? oxaloacetate (via transamination); Glu? ?-ketoglutarate (via transamination); A
Asp to fumarate via argininosuccinate has a glucogenic fate.
Asp to oxaloacetate via transamination has a glucogenic fate.
Glu to ?-ketoglutarate via transamination has a glucogenic fate.
Ala to pyruvate via transamination has a glucogenic and possibly a
Recognize that S-adenosylmethionine is a carrier of methyl groups
SAM is formed from methionine and an ATP. It is a carrier of a methyl group.
Explain that monooxygenases modify the aromatic ring using one oxygen atom from molecular oxygen (& other O atom goes to H2O)
Molecular oxygen is used to create a modified aromatic ring that has one oxygen in the product, and one oxygen ends up in water.
Explain that dioxygenases break open aromatic rings using both oxygen atoms from molecular oxygen)
Dioxygenases use molecular oxygen and incorporate both O's into the product to break open the aromatic ring. Usually used in conjunction with a monooxygenase.
Describe what is meant by cellular respiration and its three stages
Cellular respiration is the process in which cells use O2 to produce CO2 and ATP. Provides a lot of energy. Captures the energy stored in fatty acids and amino acids.
Three main stages:
1. Acetyl-CoA production
2. Acetyl-CoA oxidation
3. Oxidative phospho
Explain the general overview of oxidative phosphorylation: electrons from NADH & FADH2 being transferred down electron transport chain & pump protons; molecular oxygen is ultimate electron acceptor; energy of electron transport used to phosphorylate ADP t
The reduced cofactors of NADH and FADH2 pass their electrons to proteins in the respiratory chain. Molecular oxygen is the ultimate electron acceptor for Eukaryotes.
The energy of oxidation is used to phosphorylate ADP (Hence oxidative phosphorylation).
Summarize how the energy released by electron transport couples to the energy required for ATP synthesis: chemiosmotic theory
The phosphorylation of ADP is very thermodynamically UNfavorable. To phosphorylate, it does not use a substrate that has a higher phosphoryl potential. Energy is provided by the flow of H+'s down the gradient. The proton gradient needed can be established
Summarize & identify mitochondrial structure & their specific functions: outer membrane; intermembrane space (P side); matrix (N side); inner membrane & cisternae
Check Slides for in-depth structure/function
Outer membrane: Relatively porous membrane and allows passage of metabolites.
Intermembrane Space: Similar environment to cytosol. Has a higher [H+]. P-SIDE (Positive Side)
Inner Membrane: Relatively impermeabl
Identify electron donor (reductant, reducing agent) and electron acceptor (oxidant, oxidizing agent)
OIL RIG.
The electron donor is what is being oxidized and is the Reductant or REDUCING AGENT.
The electron acceptor is being reduced and is the Oxidant or OXIDIZING AGENT.
Explain what distinguishes E� from E�'
E� is what is actually in the real life scenario.
E�' is the standard reduction potential at pH 7 and T=25C
Use ?E�' to calculate ?G�' and vice versa
See formula. F = 96.48 kJ/(mol*V). n is moles (#) of electrons.
Apply standard reduction potentials to calculate direction of reactions
For a spontaneous reaction, the ?E�' needs to be positive. That is, E (acceptor) > E (Donor).
More positive the standard reduction potential is, the stronger the oxidizing agent (Electron acceptor); has the highest affinity for electrons.
Electrons are mo
Recognize redox centers used in the electron transport chain: FMN or FAD, cytochromes a, b & c (using Fe(II)/Fe(III)), non-heme Fe-S clusters (using Fe(II)/Fe(III)) with equal numbers of Fe & S; and number of electrons accepted/transferred at a time for e
Flavin mononucleotide (FMN) or Flavin Adenine Dinucleotide (FAD): They are the initial electron acceptors for Complex I and II. They carry two electrons and can transfer/accept ONE at a time.
Cytochromes: They carry only ONE Electron. They are an Fe-coord
Describe the two mobile electron carriers and where they are located & used: ubiquinone (Q), semiquinone radical (�QH), ubiquinol (QH2); cytochrome c
Ubiquinone is a lipid-soluble compound that readily accepts electrons. Picks up 2 electrons and protons to form ubiquinol. Ubiquinol can freely diffuse within the membrane and carry electrons and protons from one side to another. Coenzyme Q (Ubiquinone) i
Explain NADH to Complex I to Q to Complex III to cytochrome c [cyt c] to Complex IV
Pairs of electrons handed off!
NADH comes from the N side (TCA cycle and beta-oxidation) and enters complex I. FMN is reduced, which then reduces Fe-S cluster which then reduces Q (ubiquinone) to ubiquionol. Transfers electrons to complex III. Then transf
Explain FADH2 to Complex II to Q to Complex III to cytochrome c [cyt c] to Complex IV
Passes electrons individually!
FADH2 enters at complex II (TCA cycle succinate to fumarate) or from the beta-oxidation of fatty acids (which is its own thing from the arrows and such). Ubiquinone is then reduced to ubiquinol. Transfers electrons to Comple
Describe Complex I (NADH:Q oxidoreductase [know specific enzyme name]): accepts electrons from NADH on matrix side; 2 electrons to FMN which passes electrons 1 at time to series of Fe-S clusters to Q; 4 H+ pumped from matrix & 2 H+ removed from matrix to
NADH:Q oxioreductase is enzyme name!
One of largest macromolecular assemblies in mammalian cell (Nuclear and Mito. genes encode). Has an NADH binding site on the N side (Matrix). FMN is NONcovalently bound and accepts pair of e- from NADH. Then, several F
Describe Complex II (succinate dehydrogenase [know specific enzyme name]; from TCA cycle!!!): accepts electrons from FADH2
on matrix side; 2 electrons; passes electrons 1 at time to series of Fe-S clusters to Q; does NOT pump protons
Succinate Dehydrogenase is specific enzyme name! On the inner membrane of the Mito. FAD accepts two electrons, individually, from succinate. Electrons passed ONE at a time via the Fe-S centers to ubiquinone, which becomes QH2 (ubiquinol). DOES NOT pump pr
Describe Complex III (Q:cytochrome c oxidoreductase): use 2 electrons from QH2 to reduce 2 molecules of cytochrome c via Fe-S clusters, cyt b & cyt c1 via the Q cycle; Q cycle transfers 4 H+ to intermembrane space
2 electrons are used from QH2 (ubiquinol) to reduce 2 molecules of cytochrome c. Also has Fe-S clusters of cytochrome b's and c's. After the Q cycle, 4 additional H+'s are transported to intermembrane space. CHEMICAL protons NOT pumped protons!! 2 of the
Summarize the two steps of the Q cycle in Complex III: number of electrons being transferred to what molecule in each specific step & number of protons being transferred to intermembrane space or removed from matrix in each step; the net reaction for the
Chemical protons, NOT pumped protons. 2 H+'s for every QH2 molecule, hence that there is a net transfer of 4 protons per reduced Coenzyme Q. Cytochrome c can only accept ONE electron. Only uses 2 protons from matrix. See slide for better picture.
Net equa
Describe Complex IV (know this specific enzyme name: cytochrome c oxidase): 2 molecules of cyt c transfer electrons one at a time to CuA to cytochrome a to cytochrome a3; reduced Fe in cyt a3 & reduced CuB peroxide bridge from O2; 2 more cyt c molecules t
Cytochrome c oxidase is specific name!!
Contains two heme groups, a and a3, and copper ions. CuA: two ions that accept electrons from Cyt c.
CuB: bonded to heme a3 forming a binuclear center that transfers 4 electrons to Oxygen
See picture for the flow of
Describe the respirasome and its functional advantages
The respirasome is a supramolecular complex containing 2 or more ET complexes. The advantage of this is that it makes for very easy transfer of substrates and products, so everything is located in one spot to make things faster. As though it is going thro
Compare number of protons for NADH and FADH2 electron transport chains and relate this to the differences in ATP generated per NADH or FADH2�that is, the electron entry point
Complex I --> Complex IV for NADH:
NADH + 11 H+ (N-side) + 0.5 O2 --> NAD+ + 10 H+ (P-side) + H2O
Complex I --> Complex IV for FADH2:
FADH2 + 6 H+ (N-side) + 0.5 O2 --> FAD + 6 H+ (P-side) + H2O
The number of protons transported reflects the differences i
Summarize reactive oxidative species: why damaging/where generated and the roles of superoxide dismutase, glutathione peroxidase, & glutathione reductase (which uses NADPH, Ch 20)
The cause an oxidizing environment and can lead to diseases and aging. If O2 is not used properly in the ETC, the O2 can form a radical. This radical can react with protons to form O2 and H2O2 via superoxide dismutase. The hydrogen peroxide can then be us
Compare substrate-level phosphorylation with oxidative phosphorylation
Substrate-level phosphorylation uses a substrate that has a higher phosphoryl potential than ATP to phosphorylate ADP.
Oxidative phosphorylation uses the flow of protons down their electrochemical gradient to phosphorylate ADP to ATP.
Compare ATP production for glucose oxidation vs palmitate oxidation
2 ATP's are produced for the oxidation of glucose along with 2 NADH molecules.
106 molecules of ATP are produced from the oxidation of palmitate.
Compare the locations of processes occurring in oxidative phosphorylation (P side, N side, location of mito NADH & FADH2, ?-oxidation, cyto NADH, F0 and F1 of ATP synthase, ...)
ATP is created in the matrix (F1 in matrix, F0 in inner membrane). Essentially everything is in Mito. Matrix (N-side) or inner membrane. Cyt c is in the intermembrane space
Describe the two driving forces that lead to the proton-motive force
Proton-motive force is driven by:
1. Diffusion down a concentration gradient
2. Electrostatic force caused by electrical potential across the membrane. Cations go from + to -. Anions go from - to +.
Proton-motive force (delta p) = chemical gradient (pH) +
Summarize how the electrochemical proton gradient is generated by the complexes in the respiratory chain
The electrochemical gradient is created by 1 of 3 ways:
1. Actively transporting protons across the membrane (C I and C IV).
2. Chemically removing protons from the matrix by reducing Q and O2 to H2O
3. Release H+ into the intermembrane space through oxid
Explain the mechanism of coupling between electron transport & ATP synthesis
The electrochemical gradient set-up by the electron transport chain (ETC) drives the synthesis of ATP. ATP synthesis requires ETC, but the ETC also requires ATP synthesis to keep protons moving.
Interpret the effects of various inhibitors on oxidative phosphorylation if given the function of a specific inhibitor
Just know what each does to the chain and how it will inhibit. If cyanide (CN-) is added, it blocks the transfer of cytochrome c oxidase and O2; plateaus ATP production.
Venturicidin or oligomycin inhibit ATP synthase, so ATP production plateaus. DNP unco
Describe the location (sides of membrane, location in mito) and structure of ATP synthase (Complex V): F1, F0, c subunits & the relationship between the number of c subunits (number of protons transported) and ATP produced, how protons are translocated, i
ATP synthase or Complex V. Also called F1F0-ATPase if reaction is catalyzed in reverse.
F0: Integral membrane complex in inner membrane. Transports protons from intermembrane space to matrix and the energy is transferred to F1 to catalyze ADP to ATP.
F1:
Explain how Pi, ATP, & ADP are transported in & out of mitochondria
ATP Synthasome: ATP-ADP translocase and phosphate translocase are associated in membrane.
ATP-ADP transloacase: It is an antiporter that brings in ADP and spits out ATP to intermembrane space. ADP enters ONLY when ATP exits. 15% of mito membrane protein.
Summarize the glycerol 3-phosphate shuttle & why this leads to lower ATP production per NADH
Because electrons from NADH are transferred to FADH2 on inner membrane. This leads to lower ATP produced. NADH comes from glycolysis.
Summarize the malate-aspartate shuttle and its function
Only happens in heart and liver cells.
It basically takes the NADH that is found in the cytosol, and converts it to NADH that can be found in the matrix by the interconversion of malate into oxaloacetate. Takes the NADH that was in the cytosol and puts it
Explain how electrons from FADH2 from ?-oxidation of fatty acids enter the ETC
Goes from Fatty acyl-CoA to an enoyl-CoA and reduces FADH2. This FADH2 then reduces ETF-FADH2. This ETF-FADH2 the reduces Fe-S in ETF:Q Oxidoreductase. This then reduces ubiquinone to ubiquinol (QH2).
Explain the regulation of oxidative phosphorylation
Primarily regulated by substrate availability:
NADH and ADP/Pi. Due to coupling, both substrates required for ETC and ATP synthesis.
Rate of ATP synthesis controls electron flow from NADH and FADH2. Availability of NAD+ and FAD control rate of the CAC. El
Explain respiratory control
When [ADP] increases, oxidative phosphorylation rate INCREASES. Also called acceptor control.
Describe the role of uncoupling protein (UCP-1, thermogenin)
The UCP-1, or thermogenin, uncouples the ETC and ATP synthesis to generate HEAT in hibernating animals and newborns (Including humans).
Mammals, brown adipose tissue (enriched in mitochondria so it can do thermogenesis really well).