BioChem Chapter 16: Citric Acid Cycle

Only a small amount of ____ available in glucose is captured in _____

energy

glycolysis

fully ____ glucose produces a lot more energy

glucose → ____ +_____

oxidizing

6 CO2 + 6 H2O

cellular respiration:

-process in which cells consume ___ and produce ____

O2

CO2

cellular respiration:

-provides more _____ (___) from glucose than glycolysis

energy (ATP)

cellular respiration:

-_____ origin:

developed about 2.5 _____ years ago

evolutionary

billion

cellular respiration:

-what uses it? (3)

animals, plants, and many microorganisms

cellular respiration:

-occurs in three major stages:

-acetyl CoA ____

-acetyl CoA ____ in the TCA cycle

-_____ transfer and ____ phosphorylation and produce more ___

production

oxidation

electron; oxidative; ATP

Respiration: Stage 1

____-____ Production

Acetyl-CoA

Respiration: Stage 1

Acetyl-CoA production

-generates some ___, ____, and _____ (last two can be used to go into electron transport chain)

-carbohydrates release ____ of total potential _____ during stage 1

-why is acetyl CoA needed?

ATP, NADH, and FADH2

1/3; CO2

made to enter the TCA cycle

respiration: stage2 …?

Acetyl-CoA oxidation

Respiration: Stage 2

Acetyl-CoA oxidation":

-generates more ___,___, and one ___

-remaining ____ atoms from carbohydrates, amino acids, and fatty acids are ____ during stage 2

-producing ___ equivalents in form of ___ and ___-O2 needs for this to go into electron transport chain

NADH, FADH, and one GTP

carbon; releases

reducing; NADH and FADH2

(reducing e- carriers)

Respiration: Stage 3 _____ _____

oxidative phosphorylation

Respiration: Stage 3

Oxidative Respiration:

-generates vast majority of ____ during _____

-there is a ____ gradient used to make ____

ATP during catabolism

proton; ATP

in _____, stages 2 and 3 are localized to the _____

eukaryotes; mitochondria

glycolysis occurs in the?

cytoplasm

citric acid cycle occurs in the ? (except succinate dehydrogenase which is located in the ____ ____

mitochondrial matrix

inner membrane

oxidative phosphorylation occurs in the ?

inner membrane

Conversion of pyruvate to Acetyl-CoA:

-net reaction:

-oxidative phosphorylation of _____

-first carbons of glucose to be fully _____

pyruvate

oxidized

Conversion of pyruvate to Acetyl-CoA:

-catalyzed by the _____ _______ complex:

-required 5_____

-TPP, lipolysine and FAD are ____ groups

-NAD+ and CoA-SH are _____

pyruvate dehydrogenase

coenzymes

prosthetic

co-substrates

Conversion of pyruvate of Acetyl-CoA:

-very ____ reaction

-pyruvate dehydrogenase complex is ____; why we can’t use____ acids

favorable

irreversible; fatty

Structure of coenzyme A:

-coenzymes are not a _____ part of the enzyme’s structure

-they ____, fulfill a function, and _____

-the function of CoA is to accept and carry _____ groups

-CoA is a very ____ structure

permanent

associate; dissociate

acetyl

large

Structure of lipoyllysine:

-_____ groups are strongly bound to the ____

-the lipoic acid is ___ linked to the enzyme via a ____ residue

prosthetic; protein

covalently lysine

what does the lipoyllysine transfer?

electrons and acetyl groups

pyruvate dehydrogenase complex is a large _____ complex

-pyruvate dehydrogenase (___)

-dihydrolipoyl transacetylase (___)

-dihydryolipoyl dehydrogenase (___)

multienzyme

E1

E2

E3

What is the job of E1 (pyruvate dehydrogenase)?

oxidizes pyruvate to acetyl CoA

What is the job of E2 (dihydrolipoyl transacetylase)?

transfer 2-carbon acetyl groups to lipoic acid, giving acetyl CoA

What is the job of E3 (dihydryolipoyl dehydrogenase)?

regenerate cofactor lipoic acid

pyruvate dehydrogenase complex (PDC):

-advantages of multienzyme complexes…

-the short distance between _____ sites allows _____ of substrates from one ____ site to another (transfer substrate active site without leaving)

-_____ minimizes side reaction

-the _____ of the activity of one subunit affects the entire complex

catalytic; channeling; catalytic

channeling

regulation

what method was used to determine the structure of PDC?

cryoelectronmicroscopy of PDC

cryoelectronmicroscopy of PDC:

-samples are in a near-native ___ ____ state

-low ____ protects biological speciments against ____ damage

-_____ have a smaller de Brogile wavelength and produce much ___-resolution images than light

frozen hydrates

temperature; radiation

electron; high-resolution

Sequence of Events in Oxidative Decarboxylation of Pyruvate:

Enzyme 1:

-Step 1: ____ of pyruvate, bound to ___ in hydroxyl ethyl intermediate (____ is formed- product 1)

-Step 2: ____ of hydroxyl ethyl to a ____; electrons (____) reduce lipoamids (2 e- transferred) and form a ____

decarboxylation; TPP; CO2

oxidation; carbonyl; thioester

Sequence of Events in Oxidative Decarboxylation of Pyruvate:

Enzyme 2:

-Step 3: formation of ____ (product 2) (___ being reduced)

acetyl-CoA

FAD

Sequence of Events in Oxidative Decarboxylation of Pyruvate:

Enzyme 3:

-Step 4: reoxidation of the ____ cofactor

-Step 5: regeneration of the oxidized ____ cofactor; forming ____ (product 3)

lipoamide

FAD; NADH

____ is an enzyme that catalyzes a _____ reaction that does not requie a nucleotide for energy source

synthase; condensation

_____ is an enzyme that catalyzes a condensation reaction that does not require a _____ for a energy source

synthetase; nucleotide

Is the PDC reaction a part of glycolysis or TCA cycle?

NO

What are the four products of the citric acid cycle?

-NADH from dehydrogenase

-FADH2

-CO2 released

-GTP which makes ATP through substrate level phosphorylation

there are ___ acetyl-CoA per glucose

two (multiply everything by two because happens twice)

Sequence of Events in the Citric Acid Cycle:

-Step 1: C-C bond formation between ____ (2C) and ____ (4C) to make _____ (6C) - goes through ______ synthase

acetate

oxaloacetate

citrate

citrate

Sequence of Events in the Citric Acid Cycle:

-Step 2: ____ via dehydration/hydration

isomerization - sets up for decarboxylation

Sequence of Events in the Citric Acid Cycle:

-Step 5: _____ phosphprylation to give GTP (goes on to produce ___)

substrate-level

ATP

Sequence of Events in the Citric Acid Cycle:

-Step 6: dehydrogenation to give _____-

FADH2

Sequence of Events in the Citric Acid Cycle:

Step 7: ____ to add -OH group

hydration

Sequence of Events in the Citric Acid Cycle:

-Step 8: ______ to give NADH

dehydrogenation

What is dehydrogenation?

The removal of hydrogen from a molecule or oxidation of an -OH group

What is the last intermediation in TCA cycle?

oxaloacetate

what is the reaction for the C-C bond formation by condensation of acetyl CoA and oxaloacetate?

acetyl-CoA+oxaloacetate - (citrate synthase) → citrate

Citrate Synthase:

-uses ____/___ catalysis:

-carbonyl of oxaloacetate is a good _____

-methyl of acetyl-CoA is not a good _____- unless activated by _____

acid/base

electrophile

nucleophile; deprotonation

citrate synthase:

-rate ____ step of CAC

limiting

Citrate synthase:

-actively largely depends on [____]; concentration of this reactant is very low, needed for last step of CAC

oxaloacetate

Citrate Synthase:

-highly thermodynamically _____/_____; regulated by _____ availability and ____ inhibition

favorable/irreversible

substrate

product

____ fit in the citrate synthase

induced

Induced fit in the citrate synthase:

-_____ change occurs upon binding to oxaloacetate

-avoids unnecessary ____ of thioester in acetyl-CoA

conformational

hydrolysis

Induced fit in the citrate synthase:

-_____ confirmation: free enzyme does not have a ____ site for acetyl-CoA

-_____ confirmation: binding of OAA creates binding for acetyl-CoA, reactive ____ is protected

open; binding

closed; carbanion

Induced fit in the citrate synthase:

-this is an ____ mechanism

-_____ binds first and deeper in pocket

-then ____ binds

ordered

oxaloacetate

acetyl-CoA

Mechanism of citrate synthase: ______ catalysis

-_____ acts as a base and takes up a proton forming an ____ intermediate

-intermediate is stabilized by H bonding or protonation by _____

acid/base

aspartate (Asp); enol; Histidine (His)

Mechanism of Citrate synthase: hydrolysis of thioester

-____ is released from citroyl-CoA from water hydrolyzing the bond

S-CoA

Mechanism of Citrate synthase: hydrolysis of thioester

-CoA-SH goes on to be a part of the ____ reaction

PDC

Mechanism of Citrate Synthase: hydrolysis of thioester

-the thioester is _____, regenerating ___ and producing _____

hydrolyzed

CoA-SH

citrate

Isomerization by dehydration/rehydration:

-aconitase removes ____ from citrate and forms a ____ bond

alcohol

double

Isomerization by dehydration/rehydration:

-rehydration forms a ______ alcohol, more susceptible to _____

secondary

oxidation

Isomerization by dehydration/rehydration:

-what is the reactant and product of this?

citrate is reactant - (aconitase) → isocitrate (moving OH group)

Isomerization by dehydration/rehydration:

-is this a favorable reaction?

no (keep product low)

Aconitase:

-elimination of H2O from citrate gives a ___ _____ bond; hydratase

cis C=C

Aconitase:

-citrate, a _____ alcohol, is a poor substrate for ____

tertiary

oxidation

Aconitase:

-_____, a secondary alcohol, is a good substrate for _____

isocitrate

oxidation

Aconitase:

-Addition of H2O to ___-_____ is sterospecific

cis-aconitate

Aconitase:

-thermodynamically ____/_____; product concentration kept low and pull forward

unfavorable/reversible

___-___ cneter in aconitase

iron-sulfur

iron-sulfur center in aconitase:

-water removal from ____ and subsequent addition to cis-aconitate are catalyzed by the ____ center: sensitive to ____ stress

-iron-sulfur enter is coordinated by ____ residues to help bring in water hydration/dehydration; ____ can be oxidized

citrate; iron-sulfur; oxidative

Cys

Cys

Oxidative decarboxylation by _____ dehydrogenase

isocitrate

Oxidative decarboxylation by isocitrate dehydrogenase:

-isoenzymes are specific for ____ (cystolic) or ___ (mitochondrial)

-____ releases Co2

-highly ___/____ and regulated by [ATP]

NADP+; NAD+

decarboxylation

favorable/irreversible

Oxidative decarboxylation by isocitrate dehydrogenase:

-Step 1: isocitrate is _____ by hydride transfer to NAD+ or NADP+

-step 2: _____ is facilitated by electron withdrawal

-step 3: rearrangement of the ____ intermediated generates ⍶-ketoglutarate

oxidized

decarboxylation

enol

final oxidative by _____ dehydrogenase

⍶-ketoglutarate

______ dehydrogenase complex is similar to pyruvate dehydrogenase:

-same ____, identical mechanisms

-active sites ____ to accommodate different sized_____

-___ subunit is different; __ and ___ subunits are the same

⍶-ketoglutarate

coenzymes

different; substrates

E1; E2; and E3

⍶-ketoglutarate dehydrogenase:

-____ oxidative decarboxylation:

-net full ____ of all carbons of glucose

-after ___ turns of the cycle (since ___ pyruvate from glucose)

-carbons not directly from glucose because carbons lost came from ____, not ____

last

oxidation

2

2

oxaloacetate, not acetate

⍶- ketoglutarate dehydrogenase:

-___-____ is another high-energy thioester bond

-highly thermodynamically ____/___; regulated by product inhibition

succinyl-CoA

favorable/irreversible

citric acid cycle is the hub of ____

metabolism

TCA forms ___ CO2 and PDC forms ___ CO2

2

1

all CO2 generated during the citric acid cycle is produced before _____ is made

succinyl-CoA

in one turn on the citric acid cycle, neither of the carbons from _____ or ____-___ are lost

acetate or acetyl-CoA

Both ____ molecules lost were present on the ____ used to begin the cycle

CO2

oxaloacetate

generation of _____ through _____: substrate level phosphorylation by ____ synthetase; this reactio nis fairly favorable

GTP through thioester

succinyl-CoA

succinyl CoA -(_____) → succinate what else is made?

succinyl CoA synthetase GTP

Succinyl-CoA synthetase:

-___-____ phosphorylation

-energy of ____ allows for incorporation of inorganic phosphate

substrate-level

thioester

Succinyl-CoA synthetase:

-produces ___, which can be converted to ___

-____ ____ kinase catalyzes for example ADP+GTP → ATP + GDP

GTP; ATP

nucleoside dephosphate

Succinyl-CoA sythetase:

-slightly thermodynamically ____/____

-product concentration kept ___- to pull forward

favorable/reversible

low

Mechanism of Succinyl-CoA synthetase:

-has active site ___, which acts like an intermediate and accepts ____ group

-when does the substrate level phosphorylation happen?

-what is the end product?

His; phosphoryl

P from His goes GDP

succinate

oxidation of an ____ to _____ by succinate dehydrogenase

alkane to alkene

Oxidation of alkane to alkene by succinate dehydrogenase:

-oxidized ___ group to form ____ bond

-____ _____ is part of the TCA cycle and electron transport chain (complex __)

OH; double

succinate dehydrogenase

oxidation of an alkane to alkene by succinate dehydrogenase: what is the overall reaction?

succinate - succinate dehydrogenase → fumerate

succinate dehydrogenase:

-bound to ___ ____ membrane; needs to be here to grab ___ as substrate

-acts as complex ___ in the ___ ___ ___

mitochondrial inner; succinate 2; electron transport chain

succinate dehydrogenase:

-reduction of the alkane to alkene requires ___

-____ potential of carbon-_____ bond is too low for production of ___

FADH2

reduction; hydrogen; NADH

Succinate dehydrogenase:

-FAD is ____ bound, unusual

-near ____/reversible; product concentration kept ___ to pull forward

covalently

equilibrium; low

____ across a double bond: fumarase

hydration

fumerase:

_____:

-addition of water is always ___ and forms ___-malate

-OH- adds to ____- then H+ adds to the ____

-cannot distinguish between ___ carbons, so either can gain -OH

stereospecific

trans; L-malate

fumerate; carbonion

inner

Fumerase:

-slightly thermodynamically _____/_____; product concentration kept low to pull reaction forward

favorable/reversible

oxidation of ____ to a ketone and regeneration of oxaloacetate by ____ _____

alcohol

malate dehydrogenase

Malate dehydrogenase: what is the full reaction?

malate - malate dehydrogenase →oxaloacetate

malate dehydrogenase:

-creates ____

-____ step of the cycle

-regenerates ____ for citrate synthase

NADH

final

oxaloacetate