BIOC 221 - Midterm #2 (advanced editor) Flashcards
Feedforward Activation ensures that?
act in concert to overall goal of E production
An Allosteric Inhibitor does what to an enzyme?
binds to enzyme, changes its conformation and changes its substrate affinity (Km)
Bypass 1 - Reciprocal Regulation of Glucose Metabolism
Pyruvate –> ?
Pyruvate Carboxlyase vs PDH complex
Acetyl CoA
- stimulates pyruvate carboxylase (GNG)
- inhibits PDH complex (CAC)
ATP & NADH
- inhibits Acetyl-CoA from entering CAC
Reciprocal Regulation is important for two closely parallel pathways because?
direction of reaction is governed by?
it prevents concurrent activity which would waste ATP
ΔG (free energy change)
The action of an inhibitor or activator has what effect on:
a) reversible reactions
b) irreversible reactions
a) would speeds/slows reverse and forward reaction at same rate (same effect on both)
b) changes overall direction of parallel pathways
Bypass 2 - Reciprocal Regulation of Glucose Metabolism
F6P <–> F16BP
PFK-1 vs FBPase-1
- inhibited/activated by?
PFK- 1
Inhibited by: ATP, citrate
Activated by: ADP, AMP, F26BP
FBPase
inhibited by: AMP, F26BP
**Fructose-2,6-Biphosphate **
Importance? (2)
Potent allosteric regulator of PFK-1 and FBPase-1
- mediator of hormonal regulation of glycolysis and gluconeogenesis
High [F26BP] leads to?
Glycolysis increase
PFK-1 - Km decreases
Gluconeogenesis decrease
FBPase-1 - Km increases
How is cellular [F26BP] regulated?
Glucagon and Insulin
Effects of
**a) Glucagon **
**b) Insulin **
on blood [glucose]
a) raises
b) lowers
F26BP is produced under?
activates? suppresses?
formed by?
inhibited by?
normal glucose levels
PFK-1 (glycolysis)
FBPase-1 (gluconeogenesis)
PFK-2
glucagon
F26BP <—> ___
forward and reverse reaction catalyzed by?
F26BP -> F6P : FBPase-2
F6P –> F26BP : PFK-2
low blood glucose levels?
Pancreas produces Glucagon
Glucagon lowers [F26BP]
low [F26BP] leads to: PFK-1 activation & FBPase-1 inhibition
Glycolysis inhibited
Gluconeogenesis activated
Blood glucose replenished
When glucose is needed?
(4) steps
(1) Glucagon
(2) ↓[F26BP], **↑ FBPase-2, ↓PFK-2**
(3) ↓PFK-1, **↑FBPase-1 **
(4) ↑Glycolysis, ↓GNG
When glucose is in excess?
(4) steps
↑↓
(1) Insulin
(2) ↑[F26BP], ↓FBPase-2, ↑PFK-2
(3) ↑PFK-1, ↓FBPase-1
(4) ↑Glycolysis, ↓GNG
PFK-2 and FBP-2
Bifunctional protein
Glucagon(↑cAMP) - ↑ FBPase-2 (phosphorylated) - ↑GNG
Insulin - ↑ PFK-2 (OH group - dephos) - ↑ Glycolysis
PFK-2 & FBP-2
phosphate group - importance?
a phosphate group changes the shape of an enzyme and can alter substrate binding
Cellular Respiration
aerobic phase of catabolism where nutrients (sugar, FAs, aa’s) are oxidized to H2O and CO2
CAC - **localization **
glycolysis in cytosol
Pyruvate enteres mitochondria to be metabolized further by PDH and CAC
Mitochondrial Compartments
- Matrix
- Outer Membrane
- Inner Membrane Infoldings (Cristae)
`
Matrix - PDH complex, enzymes of CAC (also FA ox. and aa metabolism)
Outer Membrane - large channels (leaky)
Inner Membrane Infoldings (Cristae) - contains ETC , major permeability membrane
- contains transporters
Acetyl-CoA production from ____ by ____
Pyruvate
PDH complex
Degradation of 1 glucose to pyruvate via anaerobic glycolysis yields __ ATP.
2 ATP
Anaerobic glycolysis only yields 2 ATP.
A much higher yield can be obtained by subsequent?
complete oxidative degradation of pyruvate to CO2 and H2O by PDH complex making Acetyl-CoA, then CAC (to CO2) and then ETS
Under aerobic conditions, fate of pyruvate?
converted to acetyl-CoA and oxidized to CO2 in CAC
Pyruvate Oxidation to Acetyl-CoA and then CAC
Location?
occur in mitochondria
Since glycolysis occurs in cytosol and conversion of pyruvate to ac-CoA and CAC is in mitochondria …
pyruvate needs to be transported from cytosol to mitochondrial matrix across two mito. membranes
Inner vs Outer Membrane Transport
Inner: highly selective, has specific carrier systems for specific metabolites
Outer: non-specific pores that allows free passage of small metabolites
How does pyruvate get into mitochondria?
shuttled into mitochondria by a specific carrier system in exchange for hydroxide ion
Acetyl-CoA
- importance to metabolic pathways (specifically CAC and glycolysis)
initiator of CAC
link between glycolysis and CAC
Pyruvate → Acetyl-CoA
catalyzed by?
cofactors?
catalyzed by **Pyruvate Dehydrogenase Complex (E1 + E2 + E3) **
CoA-SH, NAD+, TPP, Lipoate, FAD
CO2 and NADH produced
IRREVERSIBLE
In vertebrates, glucose production from?
even numbered FAs impossible
Odd numbers FA’s produce propionyl-CoA (3C) which is converted to pyruvate via succinyl-CoA and OAA
PDH complex
multi enzyme complex
series of intermediates remain bound to enzyme molecules
easy flow of intermediated from one active site to another during sequential reactions **(substrate channeling) **
complex, well coordinated regulation
PDH complex - (5) coenzymes
lipoamide
Vit B1- thiamine (TPP)
B2 - riboflavin (FAD)
B3 - niacin (NAD)
B5 - pantothenic acid (part of CoA-SH)
Reactions of PDH Complex
(1)
pyruvate decarboxylated
remaining hydroxyethyl (2C) group is attached to TPP in E1
Thiamin Pyrophosphate (TPP)
derivative of?
deficiency?
derivate of thiamine (vitamin B1)
nutritional deficiency –> Beriberi (loss of neural function)
- especially affects brain which usually obtains all E from aerobic oxidation of glucose (that includes ox. of pyruvate)
Mechanism of TPP
electron sink
H+ dissociates from C between N and S to yield carbanion
e-deficient keto C of pyruvate is attacked by carbanion
then decarboxylation facilitated by e delocalization
2C hydroxyethyl group is now attached to TPP in E1
Reaction (2) of PDH complex
Hydroxyethyl (2C) group transferred to lipoamide and is concomitantly oxidized to acetyl group in E1
Swinging Arm - Lipoamide
long, flexible arm links lipoamide to E2 (core of the complex) allowing dithiol of lipoamide to swing from one active site to another
Reaction (3) of PDH complex
acetyl group is transfered from lipoamide to CoA (in E2)
at the same time, lipoamide is reduced
Reaction (4) of PDH complex
dihydrolipoamide is reoxidized to disulfide (-S-S-) form and E3-disulfide is reduced
Reaction (5) of PDH complex
the -SH group of E3 are reoxidized by mechanism in which FAD funnels 2e to NAD+ yielding NADH
FAD appears to function as an e conduit (channel)
Summary of PDH complex Reactions
Pyruvate decarboxylated & oxidized by NAD+ to acetyl in acetyl-CoA (by now 2C of glucose are lost as CO2)
Free E released during pyruvate ox. is partially stored in NADH & thioster bond in acetyl-CoA
Acetyl-CoA - central to metabolism because?
can easily donate acetate based on its high E thioester linkage
The CAC, what for?
Continuation of glucose oxidation to CO2
From 1 glucose to acetyl-CoA, we have obtained __ ATP and __ NADH during glycolysis and what from PDH rxn?
2 ATP and 2 NADH from glycolysis
2 NADH from PDH reaction
Why is acetyl-CoA the central hub of energy metabolism?
degredation of all nutrients (carbs, many aa’s and fat) comes to acetyl-CoA
The basic idea of the CAC`
releasing remaining 2 carbons (originally from glucose) in acetyl-coA as CO2 and retaining the free E in the form of ATP, NADH, FADH2
Chemistry of CAC
the 2 C in ac-CoA arent directly converted to CO2 (chemically unfeasable).
As the wheel turns, we lose 2CO2 through ox. & decarboxylation per 1 ac-CoA that enters the wheel
CAC - rxn 1
Acetyl-CoA + OAA –> citrate
In: H2O Out: CoA-SH
catalyzed by: citrate synthase
CAC - rxn 1
reaction mechanism
Citrate synthase: aldol & hydrolysis
binding of OAA to citrate synthase causes a conformation change that opens ac-CoA binding site (induced fit)
transient intermediate: citroyl-coA
citrate is a tricarboxylic acid
-∆G endergonic b/c its irreversible rxn - regulation point
Citrate Synthase Regulation
inhibited by:
- high ATP/ADP and NADH/NAD+ ratios
(high ATP and NADH indicate high E supply for cell)
- succinyl-CoA (feedback inhibition)
- citrate (product inhibition)
CAC - rxn 2
reversible hydration
Citrate –> [cis-aconitate] –> Isocitrate
H2O out then H2O in
catalyzed by: Aconitase (aconitate hydratase)
2˚ alcohol to 3˚ alcohol
(isomerization)
ENDERGONIC (+ΔG)
CAC - rxn 2
mechanism
reversible hydration
3˚ alcohol to 2˚ alcohol
+∆G: Isocitrate is quickly consumed in cell (mass action)
contains Fe-S cluster that aids rxn & binds substrate
intermediate enol compound - cis-Aconitate
CAC - rxn 2 : chemical logic
Citrate has 3 –COO– groups, which are almost fully oxidized and ready to be removed as CO2.
easiest way to lose CO2 is through ß-keto decarbox
citrate has no keto , just 1 OH
OH needs to be oxidized to keto
OH group of 3˚ alcohol cant be converted to keto so must convert to a 2˚
this step sets up for oxidation and (facile) ß-keto decarbox in following steps
CAC - rxn 3
isocitrate –> α-ketoglutarate
NAD(P)+ -> NAD(P)H + H+
Isocitrate dehydrogenase
1st oxidative decarbox. (β-keto)
enol intermediate tautomerized to α-ketoglutarate
exergonic
CAC- rxn 3
mechanism
oxidation of C2 alcohol of isocitrate w/ reduction of NAD+ to NADH
- followed by β-keto decarbox. of central carboxyl
Reaction 3 of CAC is identical to which other reaction?
6-phosphogluconate DH rxn in oxidative phase of PPP
α-KG is an important metabolite in?
amino acid metabolism
CAC - rxn 4
α-KG –> Succinyl Co-A
α-KG dehydrogenase
CoA-SH, NAD+
2nd oxidative decarbox.
exergonic
CAC - rxn 4 - mechanism
α-keto decarbox.
uses same enzymes as PDH and cofactors TP, lipoate, FAD
E released from ox. & decarb. conserved in NADH and succinyl-CoA thioester bond
By the 4rth step of the CAC, we’ve lost…
the remaining steps are to?
2 CO2
to regenerate OA to complete cycle
CAC- rxn 5
Succinyl CoA–> Succinate
GDP + Pi -> GTP + CoA-SH
Succinyl-CoA Synthetase
exergonic
CAC - rxn 5 : mechanism
synthesis of GTP
thioester cleaved driving substrate-level phosphorylation
exergonic (barely)
Pi acts as Nu allowing CoA-S to leave
Pi eventually passed to GDP to form GTP
CAC- rxn 6
Succinate –> Fumarate
Succinate DH (SDH)
prosthetic group: FAD (bound to enzyme)
ΔG ~ 0 kJ/mol
CAC - rxn 6 mechanism
enzyme-linked FAD is e acceptor (better acceptor than NAD+)
E-FADH2 is reoxidized by coenzyme-Q in ETC
this is why it is the only membrane bound enzyme in CAC
(embedded in mito. inner membrane allowing it to be part of complex II of ETC)
FeS clusters provide direct pathway for e’s to ETC leading to synthesis of approx. 1.5 ATP
CAC - rxn 7
Fumarate –> L-malate
anti-hydration (add H2O) - OH and H on opposite side
fumurate hydratase
stereospecific (only produces trans L-malate)
CAC - rxn 8
oxidation of malate (redox)
L-malate –> Oxaloacetate (regenerated!)
endergonic
Malate DH
(reduction of NAD+)
reverse rxn in gluconeogenesis (OA malate shuttle)
If Rxn 8 of CAC is endergonic, how does it occur?
driven by mass action (product depletion)
- OAA taken up quickly in cycle by highly exergonic citrate synthase rxn
[OAA] < 10-6 M makes rxn favorable
Which Rxns in CAC are irreversible?
rxn 1) Ac-CoA –> Citrate
rxn 3) Isocitrate –> α-KG
rxn 4) α-KG –> Succinyl-CoA
Prochiral
molecules that can be converted from achiral to chiral in a single step
Glucose C1 & C6 become:
CH3 of pyruvate & ac-CoA
Glucose C2 & C5 become:
Carbonyl (C=O) C of pyruvate & ac-CoA
Glucose C3 & C4 become:
lost as CO2 during PDH
If Carbonyl C is radiolabelled, at what step does the radioactivity split?
Why?
Conversion of Succinyl-CoA to Succinate
because Succinate symmetrical and the 2 COO- groups (C1 & C4) are chemically equivalent
When are radiolabelled carbonyl C lost as CO2 in CAC?`
in the second round
When is methyl (CH3) C of ac-CoA lost as CO2 in CAC?
methyl C survices 2 complete cycles but 1/2 of whats eft exits cycle on each turn after that
2CO2 that are released during 3rd round are radiolabeled
+E° ?
accepts e’s - gets reduced
-E° ?
gives e’s - gets oxidized
Products of 1 turn of CAC
3 NADH
1 ATP
1 FADH2
The Amphibolic nature of the CITRIC ACID CYCLE
metabolic pathway involved in both anabolism and catabolism
- much of CAC evolved before aerobes
- used for anabolism in anaerobes
The CAC intermediates usually remain constant as a result of?
Anaplerotic reactions that replenish CAC intermediates `
Anaplerotic reactions of the CAC (3)
1) pyruvate + HCO3- + ATP <=pyruvate carboxylase=> OAA + ADP + Pi
2) PEP + CO2 + GDP <=PEP carboxylase=> OAA + GTP
3) pyruvate + HCO3- + NAD(P)H <=malic enzyme=> malate + NAD(P)+
To keep the cell in stable steady state & to avoide wasteful overproduction, the Citric Acid Cycle is regulated by (3) ?
1) substrate availability
2) product inhibition & allosteric feedback inhibition
3) covalent modification
(4) points of CAC regulation
1) PDH complex (pyruvate –> ac-CoA)
2) citrate synthase (ac-CoA + OAA –> Citrate)
3) Isocitrate DH (Isocitrate –> a-KG)
4) a-KG DH complex (a-KG –> Succinyl-CoA)
CAC regulation by: Substrate Availability
substrate availability varies w/ cell metabolic state and [ac-CoA] & [OAA] controls citrate synthase
CAC regulation by product inhibition
a) high [NADH]/[NAD] can inhibit all Dehydrogenases by mass action & NADH competes with NAD+ for binding
b) PDH complex - ac-CoA competes with CoA for binding to E2
CAC regulation by allosteric feedback inhibition
b) Citrate Synthase & a-KG DH
- by NADH and/or ATP
CAC regulation by covalent modification (Ca2+ signals)
PDH, Isocitrate DH, a-KG DH - regulated by calcium
release of Ca2+ stored in sarcoplasmic reticulum induced by neurons (activated by Ca2+)
- contraction signal
CAC regulation by covalent modification
phosphorylation of PDH E1 by pyruvate DH kinases (PDK’s) inactivates enzyme
1) PDK’s activated by ATP (signalling excess E)
OR
2) during low [glucose], glucose required by brain so catabolism blocked in muscle mito by increase PDK activity that phosphorylates & shuts down PDH
If there is alot of citrate in mitochondria, it can be transported to cytosol, causing?
signals for FA synthesis
inhibition of: PFK-1
converted to ac-CoA & OA by citrate lyase
What happens when cells energetic needs are met?
(high [ac-CoA/citrate/ATP] favors glucose & glycogen syntheses
inhibition of CAC - accumulates ac-CoA –> FA synthesis
excess ATP inhibits Ox. phosphorylation, NADH accumulates
excess pyruvate is converted to glucose (GNG) –> glycogen synthesis
(2) hormones signal when metabolic E is required
1) Glucagon - low glucose levels
2) Epinephrine - need immediate energy
When either glucagon or epinephrine are secreted..
adenyl cyclase is activated, triggering cascade response
- cAMP acts as second messenger
- activated PKA phosphorylates lipase & perilipin
perilipin-P allows lipse-P access to lipid droplet surface
- lipase-P converts TAG’s to FA’s
transported by serum albumin to skeletal muscle, heart, kidney
enter cells by transporter
ß-oxidation to CO2 yielding ATP
the (2) degredation products of TAGs
1) free fatty acids
2) glycerol
Fate of degradative product of TAGs: Fatty acids
ß-oxidation in mitochondria (in animals)
small FAs can diffuse freely across mitochondrial membrane. How do larger FAs enter mitochondria?
Carnitine shuttle
Carnitine shuttle - (1)
activation by acyl-CoA synthetases at OMM
Carnitine shuttle (1) - activation by acyl-CoA synthetases at OMM
leaving group activation
carboxylate ion is adenylated by ATP and PPi is hydrolyzed to 2 Pi
CoA-SH thiol attacks, AMP leaves
forming fatty acyl-CoA
Carnitine Shuttle - (2)
transfer of acyl-CoA to matrix
Carnitine shuttle (2) - transfer of acyl-CoA to matrix
fatty acyl group transferred to carnitine by carnitine acyl-transferase I
transport : IMS –> matrix through acyl-carnitine transporter
fatty acyl transfer from carnitine back to CoA to regenerate fatty acyl-CoA in matrix
Why isnt fatty acyl-CoA just transported into matrix through a certain transporter?
to keep 2 seperate pools of CoA and fatty acyl-CoA (1 in mitochondria, 1 in cytosol)
- have different functions
Functions of
1) cytosolic CoA
2) mitochondrial CoA
1) biosynthetic (membrane lipids)
2) catabolic (ox. degredation of pyruvate by PDH, FAs, AAs)
Malonyl-CoA inhibits?
carnitine acyltransferase I
malonyl-CoA is 1st intermediate for FA synthesis from acetyl-CoA
- high [malonyl-CoA] indicates time for FA synthesis & inhibits entry of FAs into mitochondria
Fate of degredative product of TAG: glycerol
adipocytes lack glycerol kinase
glycerol shuttled to liver via blood & converted to G3P & DHAP for glycolysis or GNG
(3) stages of Fatty Acid Oxidation
1) oxidative conversion of 2C units into ac-CoA w/ concomitant generation of NADH
2) oxidation of ac-CoA into CO2 via CAC w/ concomitant generation of NAD+ & FADH2
3) generates ATP from NADH & FADH2 via ETC
Stage (1) - ß-oxidation
every other C is converted to C=O
allows Nu attack of CoA-SH
each round produces: 1 NADH, 1FADH2, 1 ac-CoA (2 in last round)
ß-Oxidation - step 1
dehydrogenation of alkane to alkene by acyl-CoA DH (AD) on the IMM
FAD = cofactor as e acceptor
ß-oxidation - step 2
hydration of alkene by enoyl-CoA hydratase
H2O added across double bond yields alcohol
stereospecific - only L
ß-oxidation - step 3
dehydrogenation of alcohol by ß-hydroxyacyl-CoA DH
NAD = cofactor as hydride acceptor
only L-isomers of hydroxyacyl CoA
ß-oxidation - step 4
transfer of FA chain
by acyl-CoA acetyltransferase
carbonyl C in ß-ketoacyl-CoA is electrophilic
Which 3 successive enzymes in either a pathway or cycle are analagous to 3 enzymes in ß-oxidation and why?
succinate DH
fumarase
malate DH
(oxidation of ß CH2 to alcohol then carbonyl C=O )
FA synthesis takes place in?
cytosol
FA degredation takes place in?
mitochondrial matrix
FA synthesis
FA chain elongated by 2C (acetate) units
activated donor of 2C units is (3C) malonyl-ACP
intermediates are attached to acyl carrier protein (ACP)
1st committed step in FA synthesis
formation of malonyl-CoA from ac-CoA & HCO3-
(1 ATP used)
acetyl-CoA + HCO3- –> malonyl-CoA
catalyzed by acetyl-CoA carboxylase (ACC)
FA synthesis is similar to reverse of FA degredation except (2)?
1) NADPH is used
2) stereochemistry of hydroxylated intermediate is reverse
FA synthesis: supply of acetyl-CoA
acetyl-CoA is synthesized in matric
IMM is impermeable to acetyl-CoA so acetyl-CoA units are shuttled out of matrix as citrate
shuttle also substitutes a NADPH for an NADH which is needed for synthesis
Regulation of Fatty Acid Oxidation
- *compartmentalization -**
- synthesis of TAGs* - cytosol, liver, adipocytes, intestine
oxidation to acetyl-CoA - mitochondria
rate of ß-oxidation is controlled by?
rate at which acetyl-CoA is transported into mitochondria by carnitine acyltransferase I
Regulation of FA biosynthesis (3)
1) allosteric regulation of ACC
2) regulation of gene expression by FAs
3) hormonal regulation of enzymes by covalent mod.
regulation of FA synthesis
1) allosteric regulation of acetyl-CoA carboxylase
citrate is positive effector (feedforward activation)
palmitoyl-CoA is negative effector (feedback inhibition)
Regulation of FA synthesis
3) hormonal regulation of enzymes by covalent modification (ACC)
Acetyl-CoA carboxylase
high blood glucose: insulin: activates Pase, dephosphorylates & activates ACC, malonyl-CoA inhibits ß-oxidation
low blood glucose: glucagon: activate kinase, phosphorylatres & inactivates ACC, malonyl-CoA not made, ß-oxidation to produce ATP , acetyl-CoA to CAC to make more ATP
(2) enzymes that are key to coordination of FA metabolism
1) carnititine acyltransferase 1 & acetyl-CoA carboxylase
Citrate is an effector for…
PFK-1 : inactivates
ACC: activates
How does citrate regulate?
citrate shuttle
xs mitochondrial ATP & acetyl-CoA increases transport of citrate out of mitochondria to cytosol
citrate turns down glycolysis in cytosol and switches on FA biosynthesis (increases ACC)
Malonyl-CoA as an effector
malonyl-CoA shuts down ß-oxidation
1st intermediate in FA synthesis
shuts down transport step (inhibits carnitine acyltransferase I)
good example of compartmentalization