Exam 2 (On Final) Flashcards
Metabolism (129 cards)
1
Q
What are the five principles of metabolism?
A
- Metabolic pathways: series of linked reactions that degrade fuels and construct large molecules step by step
- ATP: common energy currency, link energy-releasing & requiring pathways
- Carbon fuel oxidation: form ATP
- Types of reactions and intermediates: limited and common to many pathways
- Metabolic reactions are highly regulated
2
Q
What are the three major processes of the cell and what are the two options to get energy?
A
Energy: phototroph (sunlight), chemotroph (oxidate foodstuffs) stored as ATP
Processes
1) Mechanical work (movement)
2) Active transport
3) Synthesis (anabolism)
3
Q
What is an important general principle of metabolism pathways?
What is an important thermodynamic fact?
A
biosynthetic and degradative pathways are almost always distinct.
overall free-energy changes for a chemically coupled series of reactions is equal to the sum of the free-energy changes of the individual steps
4
Q
What are the criteria of metabolic pathways?
A
1) individual reactions must be specific
2) entire set of reactions must be thermodynamically favorable
5
Q
What is ATP composed of? What’s its activated form? What makes it energy rich?
A
Composition: adenine, ribose, triphosphate unit
Activated: complex w/ Mg2+ or Mn2+
Energy rich: because triphosphate unit has two phosphoanhydride bonds
6
Q
What happens during ATP hydrolysis?
A
energy is released as ATP is hydrolyzed to ADP and orthophosphate (Pi) and more when hydrolyzed to AMP and pyrophosphate (PPi)
- coupled to another reaction decreases AG altering the equilibrium so more product is formed
(Precise energy released depends on medium ionic strength and on Mg2+ and other metal ion concentrations)
7
Q
What are the reactions of other nucleotide triphosphates?
A
Nucleoside monophosphate kinase: NMP + ATP = NDP + ADP
Nucleoside diphosphate kinase:
NDP + ATP = NTP + ADP
8
Q
What are two important electron carriers?
A
NAD+ and FAD coenzymes and derivatives of ATP
9
Q
What is phosphoryl potential?
A
It answers the question of what phosphorylates, transfers a phosphate, well (has a stronger tendency to transfer) . Standard values are gotten from transferring a phosphate to water. (Better means more negative delta G)
10
Q
Why does ATP have a high phosphoryl potential?
A
Features of ATP’s structure. (contrast products and reactant)
1) Resonance Stabilization: Orthophosphate has greatest resonance stabilization because of its negative charges of all ATP P’s
2) Electrostatic Repulsion: Four negative charges repel–repulsion reduced with hydrolysis
3) Increase in Entropy: Now two molecules
4) Stabilization due to hydration: Water stabilizes ADP and Pi (reverse reaction now unfavorable)
11
Q
Where is ATP on the scale of phosphorylation potential? Why is this important?
A
Middle of the pack. Things like phosphoenolpyruvate, 1,2-Bisphosphoglycerate, and creatine phosphate have greater potential making ATP an excellent carrier of phosphate groups.
12
Q
What does the relative phosphorylation potentials of creatine and ATP do for the body?
A
Creatine phosphate’s greater phosphorylation potential allows for it to regenerate ATP that is quickly used up. (Prolongs energy until anaerobic metabolism takes over)
13
Q
What is one of the primary roles of catabolism?
A
ATP generation. (the principle immediate donor of free energy)
14
Q
How does the oxidation or reduction of a molecule relate to the energy storage? What’s the ultimate electron acceptor and product in carbon oxidation?
A
More reduced more energy.
- Ultimate electron acceptor in carbon oxidation is O2 and the oxidation product is CO2 (least energy)
15
Q
What is carbon-oxidation energy used for?
A
Sometimes to create ion gradient, ultimately to form ATP
16
Q
How does carbon oxidation occur?
A
Oxidation energy creates an acyl phosphate with a high phosphoryl-transfer potential (electrons are captured) which will be used to form ATP
17
Q
What do ion gradients do for cellular energy?
A
They are a form of electrochemical potential to store free energy that can make or be produced by ATP.
Oxidative phosphorylation is the formation of proton gradients by the oxidation of carbon fuels
18
Q
What’s so special about phosphates?
A
Phosphate esters are thermodynamically unstable (AG = -) and kinetically stable (stabilized by H2O, O cannot get in because of negative charge repulsion) so energy release is regulated by enzymes
(no other ions have these chemical properties)
19
Q
What are the three steps to get energy from foodstuffs?
A
1) digestion: large molecules in food broken down into smaller units
2) small molecules are degraded to a few simple units for metabolism
3) ATP produced from complete oxidation of acetyl unit of acetyl CoA
20
Q
What are the three categories of activated carriers?
A
- Of Electrons for Fuel Oxidation
- Of Electrons for Reductive Biosynthesis
- Two-Carbon Fragments
21
Q
Why are activated carriers of electrons needed for fuel oxidation? What are the two kinds?
A
O2 is the ultimate electron acceptor but the electrons do not go directly go first to special carriers
- Pyridine nucleotides or flavins: reduced forms transfer high-potential electrons to O2
Nicotinamide adenine dinucleotide (NAD+) and Flavin adenine dinucleotide (FAD)
22
Q
What are the facts of NAD+?
A
Nicotinamide adenine dinucleotide
Reactive part: nicotinamide ring (pyridine derivative)
From: vitamin niacin
Oxidation: accepts H+ and two electrons (NADH)
23
Q
What are the facts of FAD?
A
flavin adenine dinucleotide
Oxidized: FAD, Reduced: FADH2
Reactive part: isoalloxazine ring
From: vitamin riboflavin
Accepts: two protons
24
Q
What are the activated carrier of electrons for reductive biosynthesis?
A
Need high-potential electrons
Donor: NADPH (different from NADH in 2’-hydroxyl group of adenosine moiety is esterified with phosphate but carries electrons the same) Used for biosynthesis, NADH- used for ATP
25
What is the activated carrier of two-carbon fragments? Vitamin derivative? Active site? Linkage?
Coenzyme A carriers acyl groups derived from vitamin pantothenate
Active sight: terminal sulfhydryl group in CoA
Acyl groups linked by thioester bonds which are thermodynamically more unstable than oxygen ester not as stable resonance - helpful because acetyl can detach easier
26
What is the importance of activated carriers being kinetically stable?
They will not react quickly without a catalyst so enzymes control the reaction
27
How are vitamins involved in metabolism?
B vitamins are involved as biological precursors.
Other vitamins are involved in other processes in the body.
The vitamins must be modified to act as coenzymes.
28
What is the coenzyme and typical reaction type of thiamine (B1)
Thiamine pyrophosphate
Aldehyde transfer
29
What is the coenzyme and typical reaction type of riboflavin (B2)?
- Flavin adenine dinucleotide (FAD)
- Oxidation-reduction
30
What is the coenzyme and typical reaction type of Pyridoxine (B6)?
- Pyridoxal phosphate
- Group transfer to or from amino acids
31
What is the coenzyme and typical reaction of nicotinic acid (niacin) (B3)?
- Nicotinamide adenine dinucleotide (NAD+)
- oxidation-reduction
32
What is the coenzyme and typical reaction of pantothenic acid (B5)?
- Coenzyme A,
- Acyl-group transfer
33
What is the coenzyme and typical reaction of biotin (B7)?
Biotin-lysine adducts (biocytin)
- ATP-dependent carboxylation and carboxyl-group transfer
34
What is the coenzyme and typical reaction of Folic acid (B9)?
Tetrahydrofolate
- Transfer of one-carbon components; thymine synthesis
35
What is the coenzyme and typical reaction of B12?
5'-Deoxyadenosyl cobalamin
- Transfer of methyl groups; intramolecular rearrangements
36
What are the six types of metabolic reactions?
1) Oxidation-reduction: electron transfer (formation double bond/carbonyl)
2) Ligation: bond carbons using free energy from ATP hydrolysis
3) Isomerization: rearrangement of atoms within a molecule
4) Group-transfer: transfer of functional group between molecules (ie phosphorylation)
5) Hydrolytic: bond cleavage by adding water
6) Cleavage of C bond by something other than hydrolysis or oxidation: two substrates to one product (if H2O or CO2 are products double bond is formed)
37
What are the three mechanisms of metabolism regulation?
1) Alter the amount of enzyme: rate of synthesis, degradation, transcription
2) Restrict substrate availability: compartmentalism
3) Regulate catalytic activity of enzyme internally/externally: often allosteric but can be by reversible covalent modification
38
What is energy charge? How does it relate to metabolism? What is the range/the range the body keeps it in? How regulated? What are the two measurement methods?
[ATP] + 1/2[ADP] / [ATP] + [ADP] + [AMP]
- Range: 0 (all AMP) - 1 (all ATP)
- When High: inhibits ATP-generating, stimulates ATP- utilizing
- Body keeps it within 0.90-0.95 range
- Regulated allosterically by ATP/AMP
OR phosphorylation potential: [ATP]/[ADP] + [Pi]
39
What are the three destinations of pyruvate?
Ethanol, lactate, complete oxidation (CO2 + H2O)
40
How is glucose gotten from the diet?
Diet: starch & less glycogen
a-amylase (pancreatic) breaks a-1,4 bonds creating maltose and maltotriose (not a-1,6 bonds called limit dextrin)
maltase (maltose), a-glucosidase (maltotriose & oligosaccharides), a-Dextrinase (limit dextrin)
41
How does glucose enter the cell?
(location, function, Km)
Passively (no energy) via transporters GLUT1-5: have 12-transmembrane structure and distinctive roles
GLUT1 & 3: in nearly all mammalian cells (3 more in heart), basal glucose uptake, Km = 1mM (typical serum: 4-8mM)
GLUT 2: Liver, remove glucose from blood & pancreas, regulate insulin, Km = 15-20mM
GLUT 4: muscle & fat cells, uptake glucose, Km= 5mM
GLUT 5: small intestine, fructose transporter
42
What starts the steps in the stages of glycolysis?
Stage 1: Trapping and preparation
Step 1 Glucose
Step 2 Glucose 6-phosphate
Step 3 Fructose 6-phosphate
Step 4 Fructose 1,6-bisphosphate
Step 5: Dihydroxyacetone phosphate & Glyceraldehyde 3-phosphate
Stage 2: ATP Harvesting
Step 6: 1,3-Bisphosphoglycerate
Step 7: 3-Phosphoglycerate
Step 8: 2-Phosphoglycerate
Step 9: Phosphoenolpyruvate
Step 10: Pyruvate
43
What are the aspects of glycolysis step 1? Beginning Structure?
Hexokinase catalyzes a phosphoryl transfer using ATP to phosphorylate glucose to glucose 6-phosphate (G-6P)
- adding phosphate adds a negative charge at C-6 trapping ATP
Glucose: C1 U:H, D:OH, C2 U:H, D:OH, C3 U:OH, D:H C4 U:H, D:OH C5 U: CH2OH, D: H
44
What happens when glucose binds to hexokinase?
Induced fit: the two lobes move making the reaction faster:
- creating a nonpolar environment
- Water kicked out of active site increases entropy
45
What are the aspects of glycolysis step 2? Beginning structure?
Phosphoglucose isomerase catalyzes the isomerization of glucose 6-phosphate to fructose 6-phosphate (converts an aldose to a ketose)
- opening the 6-membered ring creating an enolite and the enzyme forces the enolite to the middle OH
- isomerization
- reform ring very stable not want to reopen
Glucose 6-phosphate: C1 U:H, D:OH, C2 U:H, D:OH, C3 U:OH, D:H C4 U:H, D:OH C5 U: CH2OPO3 2-, D: H
(Must happen so 2 3-C fragments will form)
46
What is an enol?
A double bond next to an alcohol in equilibrium to favor an aldehyde
47
What is the third step of glycolysis? Beginning Structure?
Phosphofructokinase (PFK) catalyzes a phosphoryl transfer using ATP to phosphorylate Fructose 6-phosphate to fructose 1,6-bisphosphate
- essentially irreversible: first big regulation
F 6-P: C2 U:CH2OH, D:OH C3 U:OH, D:H C4 U:H, D:OH, C5: U: CH2OPO3 2- D:H
48
What is the fourth step of glycolysis? Beginning and ending structures?
Aldolase catalyzes the aldol cleavage of fructose 1,6- biphosphate into glyceraldehyde 3-phosphate (GAP) and dihydroxyacetone phosphate (DHAP)
- readily reversible: good for gluconeogenesis
- GAP needed for stage 2
F-1,6-BP: C2 U:CH2OPO3 2-, D:OH C3 U:OH, D:H C4 U:H, D:OH, C5: U: CH2OPO3 2- D:H
GAP: COH C (H, OH) CH2OPO3 2-
DHAP: CH2OH CO C (H,OH) CH2OPO3 2-
49
What is the fifth stage of glycolysis?
Triose phosphate isomerase (TPI) catalyzes an isomerization (an intramolecular redox) to convert DHAP to GAP moving a double bond O from C1 to C2 through an endiol intermediate forcing double bond out
- accelerates endiol formation close to kinetically perfect
- w/o TPI would lose phosphate and produce an undesirable by-product
Mechanism: acid-base catalysis
- Glu 165 takes H from DHAP creating C1=C2 which takes a H from His 95 breaking C2=O
- Enediol intermediate: then His 95 takes H from intermediate creating O-
- GAP formed by O- forming C1=O and C1=C2 breaking, stealing H from Glu 165
- Ends stage 1
50
What is the sixth step of glycolysis? What is the structure of the product?
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) catalyzes the phosphorylation coupled to an oxidation of GAP to 1,3-bisphophoglycerate (1, 3-BPG) which has a high phosphoryl-transfer potential (Greater than ATP) as it is an acyl phosphate (mixed anhydride of phosphoric acid and carboxylic acid)
Product: 2-O3PO C=O, H C OH, CH2OPO32-
51
What are the aspects of the reaction glyceraldehyde 3-phosphate dehydrogenase catalyzes?
1) Oxidation of aldehyde to carboxylic acid by NAD+. Thermodynamically favorable
2) Joining of carboxylic acids and orthophosphate to form acyl-phosphate product. Thermodynamically unfavorable.
Coupled by an intermediate from the aldehyde oxidation linked to the enzyme by a thioester (conserving free energy released, by a covalent enzyme-bound intermediate mechanism)
52
What is the mechanism of the reaction catalyzed by glyceraldehyde 3-phosphate dehydrogenase?
Active site: Reactive Cystine, NAD+, Histidine
Step 1: hemithioacetal is formed by the reaction of aldehyde substrate with sulfhydryl on cystine
Step 2: A NADH and thioester intermediate are produced by His deprotonating the hemithioacetal so a hydride ion can be transferred to an NAD+ tightly bound to the enzyme
Step 3: The thioester is polarized to facilitate the orthophosphate attack by the positive charge on a new NAD+ replacing the NADH that leaves
Step 4: The 1,3-bisphosphoglycerate is formed and cysteine freed by the orthophosphate attacking the thioester
53
What is the seventh step of glycolysis? What is the structure of the product?
Phosphoglycerate kinase catalyzes the phosphoryl transfer from 1,3-bisphosphate acyl phosphate to ADP producing ATP (substrate-level phosphorylation) and 3-phosphoglycerate
(2 GAP => 2 ATP, replace 2 used up in first half glycolysis)
Product: -O C O, H C OH, CH2OPO3 2-
54
What is substrate-level phosphorylation?
When a phosphate donor is a substrate with high phosphoryl-transfer potential.
(in contrast with ATP formation from ionic gradients)
55
What is the eighth step of glycolysis? What is the structure of the product?
Phosphoglycerate mutase catalyzes a phosphoryl shift from 3-phosphoglycerate to 2-phosphoglycerate
Product: O C O, H C OPO3 2-, H C OH H
56
What is the mechanism of phosphoglycerate mutase?
Catalytic amounts of 2,3-bisophosphoglycerate maintain an active-site His in a phosphorylated form.
This phosphate is transferred to 3-phosphoglycerate to reform 2,3-bisphosphoglcerate which is then converted to 2-phosphoglycerate as the mutase retains the phosphoryl group to regenerate the modified His
57
What is the nineth step of glycolysis? What is the product?
Enolase catalyzes the formation of an enol phosphate phosphoenolpyruvate (PEP) by the dehydration of 2-phosphoglycerate creating a double bond
Creating a high phosphoryl-transfer potential because the phosphate traps the molecule in an unstable form
Product: -O C O, C (OPO3 2-) = CH2
58
What is the final step of glycolysis? What is the structure of the final product?
Pyruvate kinase catalyzes the (stabilizing) phosphoryl transfer from phosphoenolpyruvate to ATP producing pyruvate, a more stable ketone (this conversion drives the reaction - it is a virtually irreversible phosphate transfer)
2 ATP produced (b/c 2 GAP) are "profit"
Product: -O C O, C=O CH3
59
What is the net reaction of glycolysis?
Glucose + 2Pi + 2 ADP + 2 NAD+ --> 2 pyruvate + 2 ATP + 2 NADH + 2H+ + 2 H2O
60
What is the problem with glycolysis? The solution?
Problem: it converts NAD+ to NADH so the NAD+ needs to be replenished.
Solution: the metabolism of pyruvate (three possible reactions)
61
What are the three fates of pyruvate?
Fermentation (to ethanol or lactate) an anaerobic conditions or metabolism to CO2 and H2O in aerobic conditions
62
What is fermentation?
An ATP-generative process in which organic compounds act as both electron donors and acceptors
63
How is ethanol formed from pyruvate?
Step 1: pyruvate decarboxylase requiring coenzyme thiamine pyrophosphate decarboxylates pyruvate
Step 2: alcohol dehydrogenase catalyzes the reduction of acetaldehyde to ethanol by NADH producing NAD+
64
What is in the active site of alcohol dehydrogenase?
Zinc ion coordinated to the Sulfurs of two Cys and the N of His.
Zinc polarizes carbonyl group of substrate favoring the transfer of hydride to NADH.
65
What is the summary of the conversion from glucose to ethanol?
Glucose + 2 Pi + 2 ADP + 2 H+ --> 2 ethanol + 2 CO2 + 2 ATP + 2 H2O
- No net oxidation-reduction b/c NADH produced in first half producing 1,3-BPG from GAP is consumed in the production of ethanol
66
How is lactate formed from pyruvate?
Lactase dehydrogenase catalyzes the reduction of pyruvate by NADH to lactase
Glucose + 2 Pi + 2 ADP --> 2 lactate + 2 ATP + 2 H2O
67
How is CO2 and H2O formed from pyruvate? System? Enzyme?
Aerobically through the citric acid cycle and the ETC that transfers electrons to O2 regenerating NAD+
Pyruvate dehydrogenase complex catalyzes the oxidative decarboxylation of pyruvate to acetyl coenzyme A
68
How is fructose converted into what kind of glycolytic intermediates?
Fructose 1-phosphate pathway (mainly in the liver)
1) Fructokinase catalyzes the phosphorylation of fructose to fructose 1-phosphate
2) fructose 1-phosphate aldolase catalyzes the aldol cleavage into glyceraldehyde and dihydroxyacetone phosphate
3) triose kinase phosphorylates glyceraldehyde to glyceraldehyde 3-phosphate (glycolytic intermediate)
Other tissues: hexokinase phosphorylates fructose to fructose 6-phosphate
69
How is galactose used in the glycolytic pathway?
Galactose-glucose interconversion pathway
1) galactokinase phosphorylates galactose to galactose 1-phosphate
2) galactose 1-phosphate uridyl transferase transfers a uridyl group from uridine diphosphate glucose to galactose 1-phosphate to produce UDP-galactose and glucose 1-phosphate
3) UDP-galactose-4-epimerase then epimerizes UDP-galactose to UDP-glucose so it is not used up
4) Phosphoglucomutase isomerizes glucose 1-phosphate to glucose 6-phosphate
*UDP-glucose UDP-galactose conversion essential for galactosyl residue synthesis in complex polysaccharides and glycoproteins if too little galactose in diet
70
What happens with excess fructose consumption?
(sucrose/high fructose corn syrup)
Bypasses most important regulatory process of phosphofructokinase so creates excess Acetyl CoA which is converted to fatty acids (transported to adipose tissue and depleting ATP and Pi)
71
What causes lactose intolerance (hypolactasia)?
Deficiency of lactase that cleaves lactose into glucose and galactose leaving lactose to be consumed by microorganisms in colon fermenting it to lactic acid produces gas and human discomfort
72
What is galactosemia? Most common form?
A disruption of galactose metabolism
Most common form: inherited deficiency of galactose 1-phosphate uridyl transferase activity.
Causes: Infant failure to thrive. Vomit, diarrhea, liver enlargement--> cirrhosis, cataracts, lethargy, retarded mental development
Diagnosis: elevated blood galactose levels and galactose in urine
73
How are cataracts formed?
Aldose reductase catalyzes reduction of galactose to galactitol that is poorly metabolized and accumulates.
74
What controls the glycolytic pathway?
irreversible reactions catalyzed by hexokinase, phosphofructokinase, and pyruvate kinase
- effected by allosteric effectors or covalent modification and transcription meeting metabolic needs
- Time: allosteric- milliseconds, phosphorylation- seconds, transcription- hours
75
What is the primary control of muscle glycolysis? How?
energy charge of the cell--the ratio of ATP to AMP
-Phosphofructokinase: most important control site because committed to glycolysis (not part of any other pathway)
76
How is phosphofructokinase involved in glycolysis regulation in muscle cells?
ATP allosterically inhibits on specific regulatory site by binding and decreasing affinity (sigmoidal curve indicates slows down but does not stop completely) decreases affinity for F-6P
AMP: reverses inhibitory action of ATP (not ADP b/c 2 ADP = ATP + AMP, AMP is least in cell so most sensitive to changing concentrations)
Activity decreases when ATP/AMP ratio is lowered or glycolysis stimulated as energy charge falls
pH: a decrease in pH inhibits PFK augmenting ATP inhibition
77
How is hexokinase involved in glycolysis regulation in muscle cells?
Hexokinase is controlled by product G-6P and inhibited PFK inhibits hexokinase because the more F-6P leads to more G-6P because theses are in equilibrium
78
How is pyruvate kinase involved in glycolysis regulation in muscle cells?
Allosterically inhibited by ATP and stimulated by feedforward (substrate before enzyme activates enzyme)
79
How does the muscle at rest/during exercise effect glycolysis?
At rest: inhibits glycolysis because negative G-6P feedback and high ATP amounts
Exercise: stimulates glycolysis low charge and feed forward reaction of F-1,6BP to pyruvate kinase
80
How is liver glycolysis control different from muscle glycolysis control?
In the liver ATP and pH are not big factors (not exercising the liver) rather citrate is used.
The liver is what maintains blood-glucose level and regulates everything so the system is more complex
- Glycolysis in liver provides carbon skeleton for biosynthesis
81
How is phosphofructokinase involved in liver glycolysis regulation?
Inhibited by citrate meaning biosynthetic precursors are abundant
- Citrate enhances inhibitory effect of ATP
- Glucose abundant then F-6P abundant leads to higher F-2,6,-BP increasing PFK's affinity for F-6P: Feedforward stimulation
82
How is hexokinase involved in liver glycolysis regulation?
Hexokinase: same as in muscle
Glucokinase (isozyme): activated by high glucose levels to phosphorylate glucose - synthesizes glycogen and forms fatty acids
- inhibited by glucokinase regulatory protein
83
How is pyruvate kinase involved in liver glycolysis regulation?
L pyruvate kinase functions like M pyruvate kinase but also inhibited by alanine and controlled by phosphorylation by glucagon-triggered AMP cascade
84
How are glycolysis enzymes physically associated with one another?
Organized into complexed that increase efficiency and prevents toxic intermediated
85
What do rapidly growing cells do with glycolysis?
Such as tumor cells: metabolize glucose to lactate even in presence of oxygen
- Benefits: acidification facilitates tumor invasion, impairs immune system, reduced dependence on O2
86
What is gluconeogenesis? Where does it mainly occur? Major precursors?
The synthesis of glucose from noncarbohydrate precursors (pyruvate to glucose)
Occur: mainly in liver
(fuel for brain in RBC)
Precursors: lactate (converted to pyruvate by lactate dehydrogenase), amino acids, and glycerol
87
How do triglycerides relate to gluconeogenesis?
Triacylglycerol hydrolysis yields glycerol and fatty acids
- Animals cannot convert fatty acids to glucose, glycerol enters pathway as dihydroxyacetone phosphate
Mechanism: glycerol to glycerol phosphate via glycerol kinase then to dihydroxyacetone phosphate via glycerol phosphate dehydrogenase
88
What is the gluconeogenesis pathway? What is needed?
Pyruvate to phosphoenolpyruvate via oxaloacetate (pyruvate carboxylase then phosphoenolpyruvate carboxykinase)
Equilibrium glycolysis reactions
Fructose 1,6-bisphosphate to fructose 6-phosphate via fructose 1,6-bisphosphatase
Equilibrium glycolysis reaction
Glucose 6-phosphate to Glycose via glucose 6-phosphatase
Needs: six high phosphoryl transfer potential molecules: 4 ATP 2 GTP & 2 NADH
89
What is the first step to get pyruvate pass the irreversible pyruvate kinase reaction to phosphoenolpyruvate?
In mitochondria
pyruvate carboxylate catalyzes carboxylation of pyruvate (in three stages) to oxaloacetate using ATP
- needs biotin to activate CO2
90
How does pyruvate carboxylate function?
Tetramer of four identical subunits each with four domains:
BC: biotin carboxylate - carboxyphosphate and CO2 attachment
BCCP: biotin carboxyl carrier protein - covalently attached biotin
PC: Pyruvate carboxylase - CO2 to pyruvate forming oxaloacetate
PT: formation of tetramer activated by acetyl CoA allosteric binding
91
How does oxaloacetate become phosphoenolpyruvate?
- Malate dehydrogenase converts it to malate to get across mitochondrial membrane then reoxidized to oxaloacetate by NAD+ linked malate dehydrogenase in cytoplasm
- Phosphoenolpyruvate carboxykinase decarboxylates and phosphorylates it to phosphoenolpyruvate using GTP
92
How does gluconeogenesis overcome the PFK irreversible reaction?
Fructose 1,6-bisphosphatase hydrolyses fructose 1,6-bisphophate to fructose 6-phosphate and Pi
93
How can gluconeogensis end? Why more than one ending?
Most Tissues: end at G 6-P then to glycogen
Need glucose for blood: glucose 6-phosphatase bound to ER membrane to create glucose (occur in ER lumen)
94
How are gluconeogenesis and glycolysis regulated? The overview?
Reciprocally
Controlled by amounts and activities of enzymes: energy charge, blood-glucose concentration, glucose precursors
95
What is the first important regulation site of gluconeogenesis and glycolysis? The next one?
F 6-P and F 1,6-BP: AMP stim PFK and inhibit F 1,6- FPase
ATP &citrate inhibit PFK, stim F 1,6-FPase
pep and pyruvate in liver: ATP & ala inhibit pyruvate kinase, ADP inhib pyruvate carboxylase & PEPCK
96
How are gluconeogenesis and glycolysis regulated by blood-glucose concentration?
F 2,6-BP stim PFK inhib F 1,6-BP
Low Glucose: F 2,6-BP to F 6-P regulated by PFK2 and FBPase2 phosphorylating Ser using PKA
LBG - glucagon stim FBPase2 degrades F 2,6-BP
HBG - insulin stim PFK2 forms F 2,6-BP
Insulin & Glucagon also effect gene transcription
97
What is a substrate cycle? What is the principle? What is a futile cycle? Why are they important?
Substrate cycle: set of reaction in a loop (phosphorylation A to B hydrolysis back to A)
Principle: Not 100% on or off, they are in equilibrium. A percent change in equilibrium leads to a greater net change (net flux)
Futile cycle: substrate cycles with limited degree of cycling
- involved in pathological conditions and biologically important (metabolic signals)
Advantage: small change in rates --> large change in net flux
98
How can lactate and alanine be used? What forms them?
Lactate: formed by fast twitch muscles and RBC
- taken to cardiac/slow twitch converted to pyruvate then oxidized
- liver converted to pyruvate then glucose
Alanine: from amino acid skeletons
- liver converted to glucose
Isozymic forms of lactate dehydrogenase convert between pyruvate and lactate
99
What is the Cori cycle?
Between muscle and liver to bring lactate to the liver to convert it to pyruvate then glucose and N from amino acids to alanine
(Cycles are how things are moved in the body)
100
What converts pyruvate to acetyl CoA to start the citric acid cycle? What is the overall reaction of this step?
pyruvate dehydrogenase complex
Pyruvate + CoA + NAD+ --> acetyl CoA + CO2 + NADH + H+
101
Where do all the aspects of aerobic respiration occur?
In the mitochondrial matrix.
102
What is the overview of the citric acid cycle?
A series of oxidation-reduction reactions of acetyl group produces two carbon dioxide molecules and removes electrons from acetyl CoA oxaloacetate + acetyl --> 6C compound, release CO2 twice. 4C compound must be regenerated to oxaloacetate.
Produces (8 electrons) 3 NADH and 1 FADH2 and 1 ATP
103
What are the three enzymes and five coenzymes of pyruvate dehydrogenase complex? What are the four steps? Which is the rate limiting step?
E1: pyruvate dehydrogenase component (Prosthetic group: TPP)
E2: dihydrolipoyl transacetylase (P.g: lipoamide)
E3: dihydrolipoyl dehydrogenase (P.g: FAD)
Coenzymes: thiamine pyrophosphate (TPP), lipoic acid, FAD (catalytic cofactors) & CoA and NAD+ (stoichiometric cofactors)
1) Decarboxylation - Rate limiting step
2) Oxidation
3) Transfer to CoA
4) Regenerate enzyme
104
What is the first step catalyzed by the pyruvate dehydrogenase complex?
Decarboxylation via E1
1) **TPP** ionizes to form carbanion (H+ leaves, negative charge attack pyruvate acetyl C)
2) carbanion adds to **pyruvate** carbonyl group (H+ adds to acetyl O-, O- creates double bond breaking C1, C2 bond)
3) pyruvate decarboxylated, positive charge TPP stabilizes negative charge from decarboxylation (CO2 leaves, resonance stabilization)
4) Protonation produces **hydroxyethyl-TPP** (H+ adds)
105
What is the second step catalyzed by pyruvate dehydrogenase complex?
Oxidation by E1 (lipoamide: lipoic acid + Lys)
Hydroxyethyl group oxidizes to form acetyl group and be transferred to lipoamide forming thioester bond and reducing lipoamide disulfide group (TPP Carbanion regenerated)
106
What is the third step catalyzed by pyruvate dehydrogenase complex?
Formation of Acetyl CoA: E2
CoA + Acetyllipoamide --> Acetyl CoA + Dihydrolipoamide preserving thioester bond
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What is the fourth step catalyzed by pyruvate dehydrogenase complex?
Regeneration of lipoamide: E3
2 e- transferred to FAD than NAD+
Dihydrolipoamide + FAD --> lipoamide + FADH2 -- NAD+ --> FAD + NADH + H+
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What is the composition of pyruvate dehydrogenase complex?
Core: E2: 8 catalytic trimers form a cube. Each trimer has 3 subunits with 3 major domains. Lipoamide domain, smaller domain interacts with E3
Surrounding core: multiple copies of E1 and E3
E1: a2B2 tetramer
E3: aB dimer
Mammels: core contains E3-Binding protein (BP)
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What is the overall reaction for the citric acid cycle?
Acetyl CoA + 3 NAD+ + FAD + ADP + Pi + 2 H2O --> 2 CO2 + 3 NADH + FADH2 + ATP + 2H+ + CoA
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What is the reaction to form citrate?
Aldol condensation to citryl CoA then a hydrolysis of the thioester (powering the synthesis of a new molecule) to citrate
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What is the structure of citrate synthase? Why is this enzyme important? How does it do it?
Enzyme: homo dimer
Important: prevent side reactions of hydrolysis to acetate b/c it is a sequential, ordered reaction 1st oxaloacetate then acetyl CoA b/c oxaloacetate binding --> conformational change allowing CoA binding
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What is the mechanism of citrate synthase?
1) His 274 donates proton to carbonyl O of acetyl CoA --> remove methyl H+ from Asp 375 => enol intermediate
2) Oxaloacetate activated by His 320 H+ transfer to carbonyl C
3) acetyl CoA enol attacks Carbonyl C of oxalo. form C-C bond linking acetyl CoA and oxalo. His 274 reprotonated & Cirtyl CoA formed
4) His 274 H+ donates to hydrolyze thioester => citrate and CoA
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How is citrate converted to isocitrate? (reactions, enzyme, atoms)
Dehydration --> cis-Aconitate --> hydration
Aconitase: iron-sulfur protein
4 Fe, 4 S, 3 Cys S
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How is isocitrate converted to a-ketoglutarate?
oxidation-reduction/ oxidative decarboxylation
add NAD+ produce NADH, H+, oxalosuccinate
add H+ produce CO2 and a-ketoglutarate
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How is a-ketoglutarate converted to succinyl CoA?
oxidative decarboxylation via a-ketoglutarate dehydrogenase complex (homologous to pyruvate dehydrogenase complex)
forms thioester linkage w/ CoA has high transfer potential
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How is Succinate formed? (The Mechanism)
Cleavage of thioester succinyl CoA bond adding Pi and NDP to pick up energy producing CoA and NTP
Liver: GTP, Muscle: ATP
1) Orthophosphate displaces CoA => succinyl phosphate + CoA
2) His removes phophoryl group => succinate & phosphohistidine
3) phosphohistidine swings over to NDP
4) NTP formed with phosphate transfer
Succinyl CoA a2B2 heterodimer: succinyl CoA bound to a subunit and NDP bound to B subunit
- subunits have two domains
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What reaction allows for nucleotide triphosphates to be made from other nucleotide triphosphates?
Nucleoside diphosphokinase catalyzing
XTP + YDP = XDP + YTP
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How is fumarate formed?
A oxidation reaction. H accepted by FAD because free energy insufficient to reduce NAD+
Enzyme: succinate dehydrogenase (Fe-S): FAD isoalloxazine ring covalently attached to His
*Different*: embedded in inner mitochondrial membrane: directly associated with ETC
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How is malate formed?
hydration: stereospecific addition of H+ and OH- to form L-malate
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How is oxaloacetate formed in the CAC?
oxidation with NAD+ as the H acceptor
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How many ATP are produced per NADH or FADH2?
2.5 ATP per NADH
1.5 ATP per FADH2
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How is the citric acid cycle controlled?
- Pyruvate dehydrogenase complex (primarily) (irreversible conversion from pyruvate to acetyl CoA which is converted to CAC or lipids)
- Isocitrate dehydrogenase
- a-ketoglutarate dehydrogenase
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How does the pyruvate dehydrogenase complex control the citric acid cycle? What are the specifics for each domain?
Allosterically, phosphorylation, and hormones
- inhibited by high energy charge (@ rest)
- enhanced by low energy charge (active)
Specifically:
- ATP inhibits E1
- Acetyl CoA inhibits E2
- NADH inhibits E3
- Pyruvate and ADP enhance the enzyme
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How is E1 controlled by ATP? How do hormones effect pyruvate dehydrogenase complex?
E1
- pyruvate dehydrogenase kinase (PDK): phosphorylate E1 switch off
- pyruvate dehydrogenase phosphatase (PDP): dephosphorylate E1 switch on via hydrolysis (Add H2O get Pi) ALSO stim by Ca2+
- both associated with E2-E3-BP in mammals
Hormones
- Epinephrine binds to a-adrenergic receptor to increase Ca2+
- Insulin: stimulate phosphatase
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How is the citric acid cycle regulated at points other than pyruvate dehydrogenase complex?
Isocitrate dehydrogenase:
- inhibited: ATP and NADH
- stimulated: ADP
a-ketoglutarate dehydrogenase complex: (rate limiting step)
- inhibited: ATP, succinyl CoA, and NADH
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What are the biosynthesis intermediates from the carboxylic acid cycle?
Citrate: fatty acids, sterols
a-Ketoglutarate: Glutamate --> other amino acids--> purines
Succinyl CoA: porphyrins, heme, chlorophyll
Oxaloacetate: aspartate --> other amino acids, purines, pyrimidines OR --> glucose
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What is important about the citric acid cycle intermediates?
They must be readily replenished when used for biosynthesis
- mammals lack enzyme for net conversion of acetyl CoA into oxaloacetate or other cycle intermediate
- Oxaloacetate from pyruvate via pyruvate carboxylase
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What causes Beriberi and poisoning of mercury and arsenic do to the body?
Beriberi: neurologic, cardiovascular. Dietary deficiency of thiamine (B1)
- Mercury & arsenite: high affinity for neighboring sulfhydryl: bind to E3 and inhibit: CNS pathologies
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What in non-mammals allows for the conversion of Acetyl Coa to Glucose?
The glyoxylate cycle: isocitrate --> glyoxylate via isocitrate lyase then to Malate via malate synthase
No CO2 produced and only one NADH
