BIOC 202 Midterm 3/final

describe the overall rxn of pyruvate dehydrogenase complex (PDC)

• conversion of pyruvate to acetyl COA is catalysed by PDC and occurs in the mitochondira matrix

• invovles a decarboxylation/oxidiation of pyruvate to acetate in the form of a thioester, followed by the formation of acetyl COA

Pyruvate + NAD+ + COA-SH → Acteyl COA + NADH + CO2 + H+

what enzymes is PDC composed of?

1. E1: pyruvate dehydrogen

2. E2: dihydrolopyl transacetylase

3. E3: Dihydrolopal dehydronase

what are the 5 co-factors that compose PDC? and what enzyme subunit are they binded to?

1. Thiamine pyrophosphate (TPP) → bound to E1

2. Lipoamine → bound to E2

3. NAD+ → bound to E3

4. COA-SH (coenzyme A)→ free

describe coenyme A /COA, COA-SH in PDC

• nothing bound

• composed of ADP, pantothenate (vit. B5) and B-mercaptoethyl amine

• carrier group of acyl groups

• form higher energy thioester bonds

describe thiamine pyrophosphate (TPP) in PDC *** do we have to recongize the structure?

• derived from Vit. B1

• forms a reactive carbonion easily

• carries aldehydes

• promotes decarboxylation

describe lipoic acid (lipoamine)

• lipoicacid (black) can be attached to alysine on E2 forming a lipoamide

• Lipoamide oxidises aldehyde to acyl groups resulting in the acyl group being bound to the disulfide group

• acting as a “robotic arm”

Describe the enzyme mechanism of PDC

1. pyruvate enters E1, binds to TPP and is decarboxylated (forming O2) to form the intermediate hydroyethl-TPP

2. Lipoamide arm (oxidised) enter E1

3. The hydrozyethyl group is oxidsed to an acteyl group and is bound to the now reduced lipoamide arm (which is now called dihydrolipoyl group)

4. the reduced arm carrying the acetyl unit moves in E2 and the acetyl group is transerred to COA, forming acetyl COA (acetyl COA leaces the enzyme) is transferred to COA, forming acetyl COA (acetyl COA leaves the enzyme)

5. the reduced dihydrolopyl lipoamide arm moves into E3 where it is oxidsed by FAD (FAD is reduced to FADH2)

6. NAD+ enters E3 and reoxidises FADH2 back to the FAD (NAD+ is reduced to NADH, which leaves E# and now back at step 1)

**note: there are many copies of E1, E2, E3 enzymes and cofactors in the complex

Describe Regulation of PDC? what inhibits/activates E1, 2, 3? what enzyme?

• increase of [acetyl COA] allosterically inhibit E2

• increase o [NADH] allosterically inhibit E3

• BUT the main control is on E1, where the phosphorylation of a serine by a kinase leads to the inhibition of E2 and this the entire complex

• this kinase is called: PDC associated kinase

• Acetyl COA, NADH + ATP all stimulate the kinase, causing PDC to slow

• Pyruvate, NAD+, + ADP all inhibit the kinase resulting in the PDC gradually becoming active again

• general phosphatases will gradually (i.e. slowly) dephosphorphorylate E1, resulting in it becoming more active again

• cell signalling such as incerase in [Ca2+] +insulin activate PDC assoicated phosphatase (PDCAP) which rapidly dephosphorylates E!, leading to a rapid decrease in activity

describe kreb’s cycle

• aka the citric acid cycle, the bicarboxylic acid cycle (TCA)

• is the metabolic hub of the cell

• it completely oxidizes acetyl COA to CO2 and in the process, generates high energy e- ) in the form of NADH and FADH2) and GTP

• these e- are used in oxidative phosphorylation to generate ATP

• the kreb’s cycle is also a source of many biological precursores

• occurs in the mitochondrial matrix

describe rxn 1 of kreb’s cycle

• citrate synthase forms citrate by binding oxaloacetate to acetyl COA (going from C4 to C3)

• 2 parts:

  • 1. aldol condensation to form citryl COA

  • hydrolysis of ciryl COA to citrate and COA-SH

    • this rxn drives the entire rxn forward

• irreverisble

describe rxn 2 of kreb’s cycle

• aconitase converts citrate to isocitrate (moving the OH group)

• dehydration step to form cis-aconitate, followed by hydration step to generate isocitrate

• the delta G knot is +ve but it is drive by the -ve delta G knot of rxn 1 and rxn 2

• the OH group is moved onto the CH2 that came from oxaloactetate

describe rxn 3 of kreb’s cycle

• isocitrate is oxidised/decarboyxlated to alpha-ketoglutarate by isocitrate dehydrogenase

• generates CO2 + NADDH

• irrverible rxn

• occurs in 2 steps:

  • 1. isocitrate is oixdized to oxalosuccinate generating NADH

  • 2. Oxalosuccinate is decarboxylated to alpha-ketoglutorate (spontaneously)

note: the CO2 did not orginiate form the acetyl COA that just entered the cycle

synthase vs synthetase?

synthase: an enzyme catalyzing a synthetic in which 2 units are joined without the direct participation of ATP

synthetase: same as above BUT ATP (NTP) is directly required

describe rxn 4 of kreb’s cycle

• alpha-ketoglutarate is decarboxylated.oxidised and bound to COA by the alpha-ketoglutrate dehydrogenase complex (alpha-KGDH), generating succinyl COA, CO2 + NADH

• occurs by the same method as PDC (i.e. same 5 co-factors, similar E1 + E2, identical E3)

• back to 4C

• irreverisble rxn

• high -ve delta G knot

describe rxn 5 of kreb’s cycle

• succinyl COA synthetase converts succinyl COA to succinate generate GTP/ATP and COA-SH

• this rxn is driven by the -ve delta G knot in the cleavage of the thioester bond

**notes: -GTP can be converted to ATP by a nucleoside diphosphate kinase

-there are isoforms of succinyl COA synthetase that use ADP + generate ATP

reverible rxn

the next steps invovled in the regenrationg of oxaloacetate

describe rxn 6 of kreb’s cycle

• succinate dehydrogenase oxidises succinate, generating FADH2 and fumerate (transt)

• the free energy is not high enough to reduce NAD+

• succinate dehydronganse is part of complex II

describe rxn 7 of kreb’s cycle

• fumerase adds water across the double bond forming L-malate

describe rxn 8 of kreb’s cycle

• malate dehydrogenase oxidses malate to oxaloacetate generating NADH

• large +ve delta G knot but is driven by the 3 irreverible rxns

• also means that the matrix kep low oxaloacetate to keep the rxn moving forward

what is the next rxn of kreb’s cycle?

describe regulation of kreb’s cycle

all allosteric:

1. isocitrate dehydrogenase

   1. activiated by ADP

   2. inhibited by ATP and NADH

2. alpha-ketogluterate dehydrogenase

   1. inhibited by ATP, NADH, and succinyl COA

3. optional* citrate synthase (only in bacterica)

   1. inhibited by ATP

what is oxidative phosphorlyation?

• the formation of ATP as a result of the transfer of e- from NADH + FADH2 to O2 by electron carriers

what is electron motive force? how can it be harnessed? what does it form?

• the e- attached to NADH + FADH2 to have high transfer potential (aka EMF)

• this EMF can be harnessed by the electron transport chain (ETC) to transfer protons (H+) out of the mitochondrial matrix, through the inner mitochondrial membrane (IMM) and into the intermembrane space (IMS)

• the resulting electrochemical gradient forms a proton motive force (PMF)

  • PMF can be used by ATP syntase to form ATP (a chemical with high transfer potential)

mitochondrial composition

• the ETC and ATP synthase is located on the IMM

• the IMM is impermeable to small molecules and ions

• the OMM is permeable to small molecules and ions

• the IMM requires transporters to move things across it

• the OMM has many pores and is considered leaky

• hence, the IMS is similar to the cytosol (often referred to as the cytosol)

IMM: inner membrane of mitochondria

OMM: outer membrane of mitochondria

how can electrons be transferred?

electrons can be transferred as:

1. free e-

2. H+ atoms

3. hydride ions (H:-)

standard reductive potential

• different molecules can have different tendencies to accept e-

• this can be measured as the standard reductive potential (Eo’) in volts (v)

• the more positive the Eo’ the higher the molecules affinity for e-

what is the half rxn and coupled rxn of NAD+ and O2

• NADH + H+ → NAD+ + 2H+ + 2e-

• ½ O2 + 2e- +2H+ → H20

  ---

  NADH+ H+ + ½ O2 → NAD+ H2O → coupled half rxn

• O2 has a higher affinity for e- than NAD+

• NADH is more likely to donate e-

  • in order for e- to be transferred, the 2 rxn must be coupled

what is the nerst equation

Describe the ETC

• e- are transferred through a series of e- carriers (most of which are embedded in C1-IV) of increasing Eo’ until they reach O2 the final e- acceptor

• in the process, H+ are moved into the IMS

• the ETC is composed of 4 major complexes, each containing multiple protein subunits and e- carrier

• there are also 2 e- carriers that act as shuttles moving e- from complex to complex

describe complex 1 in ETC

• NADH-Q oxidoreductase

• accepts 2e- from NADH

• e- are transferred to FMN and then a series of 4Fe-4S clusters and finally to coenzyme Q (ubiquinone), reducing it to QH2 (ubiquinol)

• the results in 4H+ pumped out of the matrix and into the IMS

• NET RXN:

  • NADH (matrix) + 5H+ (matrix) + Q → NAD+ (Matric) + 4H+ (IMS) + QH2

describe complex II in ETC

• succinate dehydrogenase is part of this complex

• e- going from succinate to furmarate are transferred to FAD (forming FADH2), then to the succinate-Q reductase (i.e rest of CII) flowing through a series of Fe-S complexes and then finally Q (forming QH2)

  • i.e. these are the e- from FADHs of the Kreb’s cycle

• e- from NADH in CI do not pass through CII

• CII is not a proton pump so e- from FADH2 do not move as many H+ across the IMM as NADH

coenzyme Q

• Ubiquinine (Q)/ ubiquinol (QH2)

• acts as a shuttle moving e- from either CI or CII to CIII

• it is a small hydrophobic molecule located in the IMM

• contains a repeating isoprensiol tail

  • the number of repeats varies from species to species

  • e.g. in humans = 10 (i.e. CoQ10)

• ubiquinone can accept 2H+ and 2 e- to be reduced to ubiqunol

• i.e. Q+ 2H+ + 2e- ←→ QH2

complex III

• Q-cytochrome C oxidoreductase

• it contains:

  • 1× 2Fe-2S cluster

  • 2x cytochromes: cytB (contains heme bL (low affinity) + heme bH (high affinitiy))

• e- flow: QH2 → 2 Fe-2S → heme c1 → heme c

• BUT as QH2 docks, one e- follows the above, the other e- goes to heme b and participates in the Q cycle, before it follows the first e- to the 2Fe-2S cluster

• net equation:

  • QH2 + 2Cyt(ox) + 2H+ (IMM) → Q + 2CytC (red) + 4H+ (IMS)

ox = oxidsed

red = reduced

cytochrome

• e- transferring proteins containing 1 or more hemes

Q-cycle

• You do not need to know the Q-cycle expect that it is moving e- and pumping H+ through CIII

• if both e- flow through CIII, 4H+ are pumped into the IMS:

  • 2H+ come directly from the matrix

  • 2H+ come from QH2 (these H+ orignated in the matrix)

cytochrome c (cyt C)

• contains heme c

• it is another e- shuttle

• water soluble protein containing covalently linked heme

• carriers 1 e- from CIII→ CIV

• it rolls along the surface of the IMM on the IMS side

• Fe3+ + 1e- → Fe2+ (heme)

complex IV

• cytochrome C

• carriers out the final reductin of oxygen to water using e- from cyt. c

• requires 4e- to completely reduce O2 → H2O

• in the process, 4H+ are pumped into the IMS

• contains:

  • 2 cytochromes:

    • cyt a(hemea)

    • cyt a3 (hemea3)

  • 2 copper centers:

    • CuA

    • CuB

• O2 binds to heme a3 and then bridges between heme a3 + CuB

• e- flow: heme c → CuA → hemea → hemea3/CuB →O2

• net equation: 2ctyc(red) + 4H+ (IMM) + ½ O2 → 2cytc (ox) + 2H+ (IMS) + H2O

reactive oxygen species

• CIV is designed to prevent the release of partially reduced O2

ETC inhibtiors

• somes molecules can bind to various e- varriers in the ETC and block e= transfer

• e- carriers before the block will become reduced and e- carriers after the block will become oxidsed

• therefore, it blocks the ETC, prevents generation of the proton gradient of the H+ gradient, ATP synthase slows/stops, no ATP made

• need to recnogize in all forms

ATP synthase and ATP synthesis

• need to harness the PMF created by the ETC to generate ATP

• the pH difference between the matric and IMS is 1.4 pH units (~25 fold difference)

• this is done by ATP synthase

• consists of 2 components:

  • Fo

  • F1

Describe F1

• extends into the matrix + synthesis ATP when coupled to the spin generated by Fo

• contain 3 alphabeta subunits arranged in a ball

• ATP synthesis occurs in B subunit

• in the center of the ball is y (gamma) shaft

• the subunit y spins - the alphabeta subunit does not

• the y shaft binds to effect alphabeta subunit differently, causing different conformaitons in each of the 3alphabeta subuntis

describe the 3alphabeta subunits in F1

1. alphabeta lose site (L, BADP) = loading conformation

   1. ADP + Pi can bind and become trapped

2. alphabeta tight site (T, BATP) = ATP synthesis step, where ATP is made and found tightly to the B subunit

3. alphabeta open site (O, B-empty) = release confromation, it has a low affinity for ATP or ADP

• using the sping from Fo, the y shaft spins

• this causes each of the alphabeta subunits to cycle through the L,T, and O conformations

• ATP generation +release

• best estimates suggest 4 H+ need to move into the matric per ATP made

describe Fo

• embedded in IMM

• contains the half channels that H+ flow through

• uses H+ to generate spin

how does Fo cause the y shaft to spin? → only describe subunit c

• Fo is composed of many subunits, but focus on subunit c and a

• subunit c:

  • composed of 2 alpha-helices that can span the membrane

  • there are 10-12 (10 in humans) c subunits arranged in cylinder

  • the entire cylinder will rotate

  • halfway down one of the alpha-helices is a kep aspartate/aspartic acid residue which can be protonated or deprotonated depending on pH

• can only move into or be exposed to the membrane, if it uncharged (i.e. protonated) but it can move when charged if it is covered by the subunit a clamp

• the clamp masks the charge

how does Fo cause the y shaft to spin? → only describe subunit a

• Fo is composed of many subunits, but focuses on subunit c and a

• subunit a:

  • aka the clamp

  • the subunit covers 2 c subunits

  • it is stationary (i.e. does not rotate)

  • it has 2 half channels

    • 1 is open to the IMS

    • the other is open to the matrix

How does Fo use the proton gradient to spin?

1. a charged apartate subunit C is in the IMS half channel, a charged aparate subunit c is in the matric half channel

2. a proton diffuses from IMS (where [H+] is high) through the IMS half channel and protontes the aspartate to an aparatic acid

   1. now this subunit c is charged

3. the subunit c complex cylinder can rotate clockwise by unitc and freshly protnated uncharged subunit c move in the membrane

4. this brings a charged subunit c into the IMS half channel and an uncharged subint into the matrix half channel

5. a proton diffuses off the apartic acid, down the matrix half channel and into the matric

   1. this subunit c is now charged (back to step 1)

regulation of oxidative phosphorylation

• normally ATP synthesis and ETC are coupled

• ATP us formed only as fast as it is consumed

• ATP synthase cannot spin if ADP + PI are not bound to the loose alpha beta

acceptor control

• the regulation of cellular respiration by the availability of ADP as a phosphate acceptor

Describe DNP, brown fat, and FCCP

• both uncouplers

• 2,4 dinitrophenol (2,4 DNP) can uncouple the ETC and ATP synthase by carrying H+ across the IMM

• this reduces the proton gradient and the ETC speeds up to restore it

• BUT the rate of ATP synthase stays the same (if 2,4 DNP is really high is drops)

• brown fat carries thermogentics (aka uncoupling protein 1) and this is found in newborn babies and mammals to adapt to the cold

• this is a proton channel in the IMM

transport across the IMM

• electrons from NADH

  • e- transport to the ETC

  • occurs by shuttles (translocases)

Describe steps for glycerol-3-phosphate shuttle

• occurs in skeletal muscle and brain

1. cytosolic glycerol-3-phosphate dehydrogenase reduces dihydroxyactone phosphate (DHAP) to glyercol-3-phosphate oxidzing NADH → NAD +

   1. glyercol-3-phosphate is carrying the e-

2. mitochondiral glycerol-3-phosphate dehydrogenase (bound to IMM) oxidizes glycerol-3-phosphate back to DHAP in the process transferring 2e- to FAD → FADH2

3. FADH2 passes 2e- to Q reducing it to QH2

   1. QH2 → CII

   2. this is similar to CII

   3. e- by pass CI entering as FADH2

      1. therefore, use FADH2 p: o value)

describe steps for malate-asparatate shuttle

• occurs in liver, kidney, heart

1. oxaloacetate is reduced by NADH to form malate through the activity of cytosolic malate dehydrogenase (MDH)

2. Malate (carrying 2e- is transported to matrix by the malate) alpha-KG tranlocase

3. Malate is then oxdised back to oxaloactate by mitochondrial MDH, generating NADH

   1. NADH → CI BUT oxaloacetate cannotdirectly go back to the cytosol

4. oxaloacetate is transaminated by glutamate to form aspartate and alpha-KG in the matrix

5. the glutamate/aspatase translocase moves asp into the cystol alphaKG is also moved into the cytosol by mal/alphaKG translocase

6. in the reverse of 4, asp transminates alphaKG, reforming oxaloactate and Glu

   1. goes back to step 1

describe ATP, ADP, Pi as transport across the IMM

• page 41.