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84 Cards in this Set

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What is the driving step behind the tenth step of glycolysis?
Tautomerization of Pyruvate from enol --> keto form
Why bother converting Glu-6-P into Fru-6-P?
-Move carbonyl to C2
-Frees up C1 hydroxyl --> easier to phosphorylate in Fru-1,6-Bisphosphate
-Without C2 carbonyl, no reverse aldol cleavage between C3+4
What is the catalytic mechanism of Phosphoglycerate Mutase?
His at active site is already phosphorylated.

His donates P to C2 --> 2,3-Bisphosphoglycerate intermediate

His takes P away from C3, releases 2-phosphoglycerate.
What is the catalytic mechanism of aldolase?
Lys in active site forms Schiff base/imine with C2 carbonyl of F6P

Asp acts as base to catalyze aldol cleavage

G3P leaves

Asp deprotonates DHAP imine, DHAP leaves upon imine hydrolysis
What is the catalytic mechanism of GAPDH (Glyceraldehyde-3-P Dehydrogenase)?
Active site Cys attacks carbonyl C1 --> tetrahedral intermediate; NAD+ acts as base and deprotonates to allow reformation of carbonyl (acyl thioester)

SN2 rxn: Phosphate attacks C1 and displaces Cys's S

-->1,3BPG
What enzyme catalyzes the conversion of DHAP to GAP?
Triose Isomerase
What is the catalytic mechanism of Phosphoglycerate Kinase (PGK)?
Metal ion catalysis with Mg2+
What enzyme catalyzes conversion of 3PG to 2PG?
Phosphoglycerate Mutase (PGM)
What enzyme makes the first ATP in glycolysis?
PGK

Phosphoglycerate Kinase
What enzyme makes the second ATP in glycolysis?
Pyruvate Kinase

Facilitates an SN2 attack on PEP's Phosphate by O- on ADP. Displaces Pyruvate and steals its Phosphate.
What is the function of TPP?
Facilitates the decarboxylation (-CO2 lost) of pyruvate to form acetaldehyde by stabilizing the carbanion intermediate.
What structural characteristic is shared by most allosterically regulated enzymes?
They are multimers: multi-subunit proteins.
Which steps are regulated in glycolysis?
Step 1, 3, 10
How is hexokinase regulated?
Inhibited by G6P: product negative feedback loop.

PFK also talks to Hexokinase: if it stops it causes G6P to accumulate and slow Hexokinase too.
How is PFK regulated?
Allosteric activators: AMP, Fru-2,6-Bisphosphate (R state)

Allosteric Inhibitors: citrate, ATP (T state)

Active site Arg 162 in R state attracts F6P
Changes to Glu 161 in T state --> repels F6P.
What happens to enzyme activity curves upon addition of allosteric activators?
They become more hyperbolic as enzyme activity increases.
What happens to enzyme activity curves upon addition of allosteric inhibitors?
They become more sigmoidal in shape.
What is the advantage to regulating enzymes in both the forward and reverse reactions in a pathway?
Activating forward + inhibiting reverse = larger effect than altering the rate of just one.
What is the principle behind global regulation?
Tissues have isozymes (different enzyme but same reaction) with different affinities and speeds --> different rates of flux.
What is the principle behind allosteric modification?
Enzyme in T/R state equilibrium.

Effector molecules bind away from active site.

Inhibitor --> T state equilibrium shift
Activator --> R state
What enzyme cofactor is involved in ALL a-keto acid decarboxylation reactions?
Thiamine Pyrophosphate, TPP.

Thiazolium ring --> carbanion upon deprotonation; nucleophilic attack on alpha-carbonyl to allow decarboxylation.
What are the major regulatory enzymes in the TCA cycle?
Step 1: Citrate Synthase

Step 3: Isocitrate Dehydrogenase

Step 4: alpha-ketoglutarate Dehydrogenase
What is the net yield of
NADH
FADH2
CO2
GTP
per mole of pyruvate?
3 NADH
2 CO2
1 FADH
1 GTP
What enzyme catalyzes the "linking step" reaction?
Pyruvate Dehydrogenase Complex

pyruvate + coA + NAD+ --> acetyl-coA + CO2 + NADH + H+
What reaction does pyruvate dehydrogenase catalyze?
TPP-mediated decarboxylation of Pyruvate, transfer to Lipoic Acid tail of E2
What reaction does Transacetylase catalyze?
Oxidation of hydroxyethyl group to acetyl group; transfer to coA
What reaction does Dihydrolipoyl Dehydrogenase catalyze?
Reoxidation of lipoic acid by FAD, reduction of NAD+ to NADH2. Restores E2 so it can begin another catalytic cycle.
How is PDH regulated?
Kinase Phosphorylates PDH --> inactivates

Phosphatase dephos. PDH --> activates

PDH inhibited by NADH, Acetyl-CoA
How is PDH Kinase regulated?
HIF-1 (upregulated due to hypoxia)

PDH Substrates inhibit: ADP, Ca2+, NAD+, CoA-SH

PDH products activate: ATP, NADH, Acetyl-CoA
How is PDH Phosphatase regulated?
Activated by Ca2+, Mg2+ which indicate muscle contraction and need for ATP.
What is purpose of TCA cycle?
Make reducing power for Electron Transport Chain
Oxaloacetate + Acetyl-CoA = ?
Citrate Synthase --> Citrate
Citrate --> ?
Aconitase --> Isocitrate
Isocitrate --> ?
Isocitrate Dehydrogenase --> CO2 + NADH + a-ketoglutarate

decarboxylation at C3
a-ketoglutarate --> ?
+ NAD+ + CoA-SH --(a-ketoglutarate dehydrogenase)--> NADH + CO2 + Succinyl-CoA

Decarboxylation at C5

CoA-SH reduces NADH
Succinyl-CoA --> ?
+ GDP + P --(Succinyl-CoA Synthetase)--> GTP + Succinate + CoA-SH
Succinate --> ?
Succinate Dehydrogenase + FAD+ --> FADH2 + Fumarate

Oxidation: Succinate loses 2 H's and forms C2-C3 double bond
Fumarate --> ?
Fumarase + H2O --> Malate

hydration of double bond
Malate --> ?
Malate Dehydrogenase + NAD+ --> Oxaloacetate + NADH

oxidation of hydroxyl
Glycolysis

Glucose --> ?
Hexokinase +ATP --> Glucose-6-Phosphate + ADP
Glycolysis

Glucose-6-P --> ?
Posphoglucose Isomerase --> Fructose-6-P
Glycolysis

Fructose-6-P --> ?
Phosphofructokinase + ATP--> Fructose-1,6-Bisphosphate + ADP
Glycolysis

Fructose-1,6-BP --> ?
Aldolase --> Dihydroxyacetone Phosphate/DHAP + Glyceraldehyde-3-Phosphate/GAP
Glycolysis

DHAP --> ?
Triose Isomerase --> GAP
Glycolysis

GAP --> ?
Glyceraldehyde-3-Phosphate Dehydrogenase + P + NAD+ --> 1,3-Bisphosphoglycerate + NADH
Glycolysis

1,3-Bisphosphoglycerate --> ?
Phosphoglycerate Kinase + ADP --> 3-PG + ATP
Glycolysis

3-Phosphoglycerate --> ?
Phosphoglycerate Mutase --> 2-Phosphoglycerate

2,3-PG intermediate, His-mediated P transfer
Glycolysis

2-Phosphoglycerate --> ?
Enolase --> Phosphoenol Pyruvate + H2O

dehydration
Glycolysis

PEP --> ?
Pyruvate Kinase + ADP --> Pyruvate + ATP

Pyruvate: enol --> --> KETO form
What molecule controls the overall balance of the TCA cycle?
NADH

Controls both citrate synthase and a-ketoglutarate dehydrogenase
Why does HIF-1 respond to hypoxic conditions by upregulating PDH Kinase?
PDH Kinase shuts off PDH, which is the "gateway" to the AEROBIC metabolic pathways, which are useless in low oxygen conditions.
What is the structure of lactose?
B-1,4 Galactose + Glucose
What is the structure of sucrose?
A-1, B-4 Glucose + Sucrose
What is the structure of amylose?
Long A-1,4 Glucose chains

Forms helical structure
What is the structure of amylopectin?
Amylose with branches

Long A-1,4 glucose chains
occasional A-1,6 glucose branches
every 24-30 glucoses
What is the structure of cellulose?
Long B-1,4 glucose chains
What is the structure of glycogen?
Amylopectin with MORE branches

Long A-1,4 glucose chains
occasional A-1,6 glucose branches
every 12 glucoses
What is the structure of NAD?
2 x ribose 5-phosphate
joined by phosphpdiester bond

one ribose: adenine residue

other one: nicotinamide residue, e- acceptor
What is the structure of FAD?
Ribose-5-PP + adenine residue

Riboflavin ring attached to PP via phosphoester bond.
http://wps.prenhall.com/esm_horton_biochemistry_4/37/9594/2456230.cw/content/index.html
great tca cycle quiz
What is the reaction mechanism of succinyl-coA synthetase?
removal of S-CoA from succinyl coA through His-mediated attack on carbonyl by Pi

Pi displaces S-CoA; succinate and S-CoA both released; PO3- attached to His

His-mediated phosphorylation of GDP
Which reactions in the TCA cycle generate CO2?
isocitrate + NAD+ --(isocitrate dehydrogenase)--> a-ketoglutarate + CO2 + NADH

a-ketoglutarate + NAD+ + CoA-SH --(a-ketoglutarate dehydrogenase)--> succinyl CoA + CO2 + NADH
Which reactions in the TCA cycle generate FADH2?
Succinate + FAD+ --(Succinate Dehydrogenase)--> Fumarate + FADH2
Which reactions in the TCA cycle generate NADH?
isocitrate + NAD+ --(isocitrate dehydrogenase)--> a-ketoglutarate + CO2 + NADH

a-ketoglutarate + NAD+ + CoA-SH --(a-ketoglutarate dehydrogenase)--> succinyl CoA + CO2 + NADH

malate + NAD+ --(malate dehydrogenase)--> oxaloacetate + NADH
What is an anapleurotic pathway?
rxn that feeds intermediates into a system.

replenishes
What is a catapleurotic pathway?
rxn that siphons intermediates away from the system.

depletes
What is an amphibolic pathway?
A pathway that both builds larger molecules and breaks others down
What is the purpose of the Malate-Aspartate shuttle? What steps are involved?
Transfer reducing power from mitochondrial intermembrane space to the inner matrix.

Malate + cMDH --> oxaloacetate --> aspartate

aspartate passes into matrix

aspartate --> oxaloacetate --> mMDH --> malate

malate passes back into cytoplasm.
What does antimycin A block in the ETchain?
Transfer of e- from cytochrome b to cytochrome c1 within the Complex III protein.
Which complexes pump protons out of the mitochondrial matrix?
Complexes I, III, IV
What is the second source of electrons for the ETchain, aside from NADH?
Succinate --> Fumarate + 2e- + 2H+ in Complex II.

These electrons are carried by FADH2 to Ubiquinone.
Where does amytal block in the ETChain?
First step: NADH donating e- to complex I
Where does rotenone block in the ETChain?
First step: NADH donating e- to Complex I
Where does cyanide block in the ETChain?
Very last step: passing of e- from Complex IV to O2
How can you circumvent addition of amytal or rotenone to the ETChain?
Add succinate. This bypasses the block and supplies e- through Complex II.
What is the effect of adding 2,4 Dinitrophenol to the cell?
It dissipates the H+ gradient

e- transport continues, O2 consumption continues

no ATP synthesis occurs.
How does Triose Phosphate Isomerase (TIM) drive the DHAP --> GAP reaction forward?
Binds intermediate 150x better than it binds DHAP --> rxn goes forward.
What is the anaerobic fate of pyruvate in muscle cells?
Pyruvate + NADH --(lactate dehydrogenase)--> lactate + NAD+ + H+
What is the anaerobic fate of pyruvate in yeast cells?
pyruvate --(pyruvate decarboxylase/TPP)--> acetataldehyde + CO2

acetaldehyde + NADH --> etOH + NAD+
Why does alanine inhibit Pyruvate Kinase?
Ala can easily be turned into pyruvate --> no need to make pyruvate from PEP.
How is pyruvate kinase regulated?
Phosphorylate to deactivate

inhibited by ATP, alanine

STRONGLY activated by Fructose2,6-bisphosphate, AMP
How do oligomycin and venturicidin affect the electron transport chain?
They stop it because they prevent H+ gradient dissipation.

Eventually no more e- can flow because gradient is too high.
What causes the conformational change in the a/b subunits of F1 ATPase?
Gamma subunit rotates with F0 intermembrane domain "proton turbine."

Which a/b dimer the gamma subunit contacts determines conformation.
Describe the reactions in the glycerophosphate shuttle.
DHAP + NADH --> glycerol-3-phosphate

glycerol 3-phosphate + FAD+ --> DHAP + FADH2 in membrane

FADH2 feeds e- to Complex II in ETChain.