biochem exam 3

8/10/2019 Biochem Exam 3

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Biochem Exam 3 11/6/2014 11:22:00 AM

Multiple Fates of Pyruvate

Fermentation: regen NAD+; remove pyruvate

o No energy gain; toxic accumulation

Respiration

o

Slow; LOTS of ATP; CO2 NAD+/NADH role catalyzes hydride transfer

Glycolysis Regulation: Allostery

ATP high; low glycoloysis

ATP low; high glycolysis

Feedback Inhibition

o Downstream products inhibit PFK

Liver in Process

LDH: pyruvate to lactate which is moved to liver

----------------------------------End of Exam 2-----------------------------------

Gluconeogenesis Begins

Previous lectures

Anaerobic conditions: muscles produce lactate from pyruvate

Yield: 2 ATP

Cori Cycle

Regeneration of glucose from lactate and protons;

gluconeogenesis

Mostly occurs in liverGluconeogenesis

Not a reversal of glycolysis; remember that there are 3 irreversible

steps which need to be circumvented; other 7 steps are simple

reversal

Rather than reversal: PyruvatePEP; Fructose-1,6-

bisphospahteFructose-6-phosphate; Glucose-6-

phopshateglucose

Energy CONSUMING process

Anabolic process (synthesis)

PyruvatePEP

PEP is a higher energy molecule than pyruvate; have to hydrolyze

ATP to get it there

PyruvateoxaloacetatePEP


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Pyraveoxaloacetate = ATP hydrolysis (~7kcal/mole); pyruvate

carboxylase

OxaloacetatePEP = GTP hydrolysis (~7kcal/mol); PEPCK

Happens in mitochondria

CO2 is reproduced (consumed first and remade with making of PEP) STEP 1

o Biotin cofactor in attaching CO2 to pyruvate to form

oxaloacetate

o Pyruvate carboxylase

o Bicarbonate rips phosphate from ATP and reacts with biotin-

enzyme complex to form CO2-biotin-enzyme complex (first

two steps)

o That complex reacts with pyruvate through substitution to

yield biotin-enzyme + oxaloacetate

Biotin

o Usually found as a protein conjugate; concerned biotin reacts

w/ activated CO2

Mitochondria

Inner membrane highly selective; oxaloacetate CANNOT move in

and out

Oxaloacetate is taken out in the form of malate via transport

system and reoxidized to oxaloacetate in cytosol (carbonyl carbonof oxaloacetate is reduced to OH- to form malate)

Glucose-6-phosphate to glucose happens in endoplasmic reticulum;

irreversible step

G6P converted to glucose + Pi by glucose-6-phosphatase

SP = Ca2+ protein necessary for phosphatase activity

T1 = transport G6P into ER Lumen

T2 (Pi) and T3 (glucose) to transport products into cytoplasm

Glucose-6-phosphatase and SP for reaction

Final Thoughts: glycolysis and fermentation happen completely in cytosol;

but gluconeogenesis requires multiple organelles


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Lipids 11/6/2014 11:22:00 AM

Gluconeogenesis: non-carb to glucose; ATP consumption (2 pyruvate->1

glucose)

Oxaloacetate transportation happens by converting it to malate;

o Oxaloacetate (mit.) + NADH (mit.) + H+ (mit.) + NAD+

(cyt.) Oxaloacetate (cyt.) + NAD+ (mitoc.) + NADH (cyt.)+ H+(cyt.)

1 Glucose = 6 ATP consumed through gluconeogenesis

Can use pyruvate, lactate, amino acids, and glycerol

Using glycerol, it costs 1 ATP to convert to Glyceraldehyde-3-P and

to glucose (2 per two molecules of glycerol, etc)

Net energy loss (2 ATP by glycolysis; -6 by gluconeogenesis)

LactatePyruvateGlucose (liver)

Reciprocal regulation of glycolysis and gluconeogenesis

ATP stops glycolysis by inhibiting PFK

Fructose-2,6-bisphosphate: promotes glycolysis; inhibits

gluconeogenesis by inhibiting fructose-1,6-bisphosphatase;

activates PFK promoting glycolysis


4/23

Blood Glucose dropsPKAPhosphorylase kinasephosphorylates glycogen

phosphorylase

--------------------------------Lipids Begins-------------------------------------

Lipids-hydrophobic; amphiphilic small molecules

Fatty acids = hydrocarbon chain + carboxylic acidn = linear chain (no branches)

cis-9 = double bond between C9 and C10 and it is in cis

Section 12.2 Berg

Lipids = water insoluble biomolecules; highly soluble in organic

Phospholipids; glycolipids; cholesterol

Ester connects fatty acid to glycerol

Phospholipid = phosphate to alcohol; both to glycerol and fatty acid

chains attached to glycerol

Glycolipids = contains sugar molecules; usually contain sphingosine

in animals

Cholesterol = multiple (4) rings; steroid nucleus

Phospholipids

Have polar and non-polar regions

Several different polar head groups for phospholipids

Glycerolipids

Weird; have sugar heads that are hydrophilic

Cholesterol Hydrophobic

More rigid than phospholipids; four rings

Membrane Proteins

Integral inside the membrane itself

Peripheral usually water soluble so they are attached to the

membrane

o Attach hydrophobic groups to protein and send it into the

membrane (GPI linkage)

o Often found in extracellular proteins

Cholesterol

o More cholesterol = less fluidic

Proteins and lipids move laterally

Lipids can FLIP

Membrane is heterogeneous and dynamic (changes over time and space)


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Lipid Rafts are a thing

Signaling proteins enriched in cholesterol rich subdomains

Differ from regular bilayer due to enriched area


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Mitochondrial Metabolism 11/6/2014 11:22:00 AM

Pyruvate Dehydrogenase links glycolysis to TCA

PDH

o Mitochondrial enzyme complex

o Transfers a hydride to NAD+ to form NADH

o

Net Rxn: Pyruvate + CoA + NAD+ acetyl-CoA + CO2 +NADH + H+

Points to remember about PDH

o PDH highly regulated

o Made of multiple copies of 3 Enzymes (E1, E2, E3)

o Uses severalcofactors (TPP, lipoic acid)

o Rxn intermediates delivered to next active site covalently

o Removes carbon from Pyruvate

o Generates NADH

o CleavesPyruvate between C2 and C3 (between carbonyl

carbons), leaving the carbon w/ 2 oxygens to become CO2

and the other to become acetyl CoA

o Be able to answer which carbons in glucose become

carbon dioxide

o

CoA

Vitamin B5 Derived Cofactor

Non-protein required for enzymesCoA-SH

Thiol group is very important to function

Takes carboxylic acid and attaches to something else

CoA-SH + RCO2H CoA-carboxylic acid + H20

Used as an Acyl carrier

Acetyl CoA

Roles: TCA; Fatty Acid Synthesis; Cholesterol Synthesis; histone

acetylation (regulatory roles)

After formation of acetyl-CoA, it immediately remains in the

mitochondrial matrix

Reacts w/ oxaloacetate to form citrate through citrate synthase

Oxaloacetate + Acetyl CoA + water citrate + CoA

In this way, CoA is reformed to become Acetyl CoA again

Citrate


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Generated from oxaloacetate and acetyl CoA through citrate

synthase

Can be diverted to generate cytosolic acetyl CoA mammals

Forms cis-Aconitate and then form isocitrate; through Aconitase

Isocitrate Dehydrogenase (IDH) Generates NADH and CO2

Forms -ketglutarate

o Important for amino acid synthesis and other processes

Genes (IDH1/2) are most frequently mutated in human cancer

In cancer, 2-hydroxyglutarate is created from the mutated gene,

which stimulates cell growth

-ketglutarate becomes Succinyl Co-A through interaction of CoA and NAD+;

yielding CO2 and NADH

Succinyl-CoA is then converted to Succinate, yielding ATP or GTP

SuccinateFumarateMalate>OAA

First transformation requires FADFADH2

FAD (Flavin Adenine Dinucleotide)

Different from NAD+

Another cofactor that causes oxidoreduction roles in cells

Reduces to FADH2

TCA Cycle

Acetyl CoA + 3NAD+ + FAD + ADP + Pi + H20 2CO2 + CoA + 3NADH + FADH2 + ATP + 2H+

Net yield: 2CO2 + ATP + 3NADH + 1 FADH2

Pyruvate into Citric Acid cycle = 4 NADH (1 from conversion of

pyruvate to Acetyl CoA)

PDH Regulation

Inhibit PDH by phosphorylating it

PDH Kinase

TCA regulated in irreversible steps (pyruvate to Acetyl CoA, Isocitrate to

ketoglutarate, ketoglutarate to succinyl CoA)

Energy Charge = (ATP + 0.5ADP/ ATP + ADP + AMP)

TCA interaction w/ other pathways

Pyruvate carboxylase makes oxaloacetate from pyruvate

(gluconeogenesis)

Fatty acids can be degraded to Acetyl-CoA


8/23

Glutamine can be converted to Glutamate and converted to

ketoglutarate to feed TCA cycle


9/23

Oxidative Phosphorylation 11/6/2014 11:22:00 AM

Summary of TCA

1 ATP; 3 NADH; 1 FADH2 + 2CO2

Electron Transport Chain (ETC)

NADH and FADH2 in mitoc. oxidized to NAD+ and FAD

Electrons serially transferred to O2 to become H20 Mitochondrial membrane

Protons pumped out

Four Complexes; 3 pump protons (not Complex II)

NADH Reductase (Complex I)

o Oxidizes NADH to form NAD+

o 2 electrons and 2 H+ from NADH and delivered to Q

o 4 H+ moved to intermembrane space

Succinate Dehydrogenase (Complex II)

o Two electrons and 2 H+ from FADH2 and delivers them to Q

o Does NOT pump H+ to intermembrane space

o FADH2 not as stable as NADH so the electrons and protons

are immediately transferred to coenzyme Q after FADH2

generation

Coenzyme Q = ubiquinone

o Quinone compound (2,5 cyclohexene w/ two oxygens)

o Membrane soluble

o

Takes 2 electrons and 2 protons to form QH2 QH2 delivers electrons to Complex III (cytochrome C reductase)

o Q cycle- complex III pumps protons; relays electrons to

cytochrome C

Complex III has two Q binding sites (Qo and Qi)

Cytochrome C binds to outer side of complex III

Oxidized Q binds to Qi site

Reduced QH2 from Complex I or II comes to Qo site

QH2 gives one electron to cyt C; one electron to Q in Qi

site and 2 protons to outside

Oxidized Q moves out of complex and another QH2

enters Qo site

4H+ to intermembrane space

Cytochrome C

o Water soluble (small molecule) protein w/ a heme


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o Takes one electron each time from QH2 and delivers it to

Complex IV

o Works twice per QH2

Complex IV

o

Moves elects from cytochrome C ot 1/2O2, moving 2 moreprotons to intermembrane space

Overall assume, 2.5 and 1.5 molecules ATP from each mitochondrial

NADH and FADH2 molecule

Problem of lonesome NADH from glycolysis

Cannot enter mitochondrial matrix to enter ETC

So how much ATP value does this have?

o Electrons from NADH are transported across mitochondrial

membrane via shuttles

o G3P Shuttle ATP = FADH2 power

NADH reduces DHAP to G3P in cytoplasm

G3P Reduces FAD to FADH2 which is oxidized to FAD

making QH2 from periphery

o Malate-asparatate shuttle = mitochondrial NADH power

Malate to aspartate inside w/ production of NADH

Aspartate to malate outside w/ production of NAD+

Only works if NADH/NAD+ ration is higher in cytosol

than matrixF1 F0 ATP Synthase

Mitochondrial ATP synthase

F0 transports H+ from outside to inside; unidirectional rotation

F1 uses rotational energy to produce ATP from ADP + Pi

F1 Subunit

Composed of 9 subunits

Alpha3, Beta3, Gamma, Delta, epsilon

Alpha and Beta are hexamers; both bind nucleotide, but only B

participate in reaction

Beta binds tightly to ATP bringing the energy level down below ADP

Loose conformation binds ADP and Pi; second Beta converts to ATP;

Third releases

Gamma rotation causes change in conformation allowing release

F0 subunit


11/23

a subunit composed of hydrophobic outside and hydrophilic half-

channels

c subunit has aspartic acid in middle of transmembrane helix

when aspartic acid residue is in membrane, it will be protonated

when exposed to polar environments, it will be de-protonatedF1F0 Summary

Proton from inter-membrane space to matrix

Rotates c subunit of F0

Rotates Gamma of F1

B conformation changes

ATP synthesized from ADP and Pi


12/23


13/23

NADPH comes in with a proton and oxidized to NADP+ to make

alcohol and add a hydrogen to second to last carbon

NADPH

NADPH used in major anabolic processes

Anti-oxidant against oxidative stress in cells Used to produce reactive oxygen in immune cells to kill

microorganisms

Completely reduces second half of the malonyl ACP to a

hydrocarbon looking thing

Releases water when OH leaves

Each cycle generates 2 more carbon attachments; 2 NADPH

consumed; cleave results in fatty acids

Fatty acids attached at OH and Phosphate group of Glycerol-3-phosphate

Phospholipids

De Novo Synthesis overridden when theres a ton of fatty stuff in your food

Fatty Acid Oxidation (beta oxidation)

Mitochondrial process

Generates CoA (some ATP) from fatty acids

Cyclic process

Each cyle removes 2 carbons from carboxyl ends and taken out and

become acetyl CoA

Each produces 1 FADH2 and 1 NADH and 1 Acetyl CoA, but at theend there will be an extra Acetyl CoA remaining so it/s always 1+

(number of NADH) = number of acetyl CoA from molecule

Fatty acids for energy

Triacylglycerol cleaved into fatty acids and glycerol

Fatty acids fatty acid coenzyme A and move into mitochondria

Fatty acid CoA cannot enter directly

o Uses Carnitine shuttle

o Coverted to acyl carnitine and enters mitochondria

o Inhibited by malonyl CoA

o High levels of NADH in mitochondria also inhibits fatty acid

translocalization


14/23

Amino Acid Metabolism 11/6/2014 11:22:00 AM

If there isnt enough oxaloacetate, there will not be anything for TCA cycle,

leaving extra acetyl CoA

This is converted to ketone bodies which are used by other organs

for energy supply

Some diseases have nail-polish remover like-smellCholesterol Synthesis

Same de novo vs. food salvage process

-----------------------------End----------------------------------------------------

Amino Acid Metabolism

Nitrogen Fixation

N2 to NH3 by some bacteria

Nodules in rhizobium (soil bacteria) form reaction: N2 + 3H2

2NH3

2/3 nitrogen fixation

Ammonia reacts with ketoglutarate to form glutamate, consumes one NADH

or NADPH

Goes from achiral (ketoglutarate) to chiral (glutamate)

Sometimes Glutamate becomes glutamine by interacting with another

ammonia

Amine is donated from glutamate/glutamine and regenerate their original

molecules (glutarate or glutamine)

Pyridoxal Phosphate: vitamin derived cofactor that transfers ammonia andthen switches between ketone to amine or amine to ketone

Stereospecific

Generates L-amino acids (S-form)

Amino Acid Degradation

First step is to give up an amine to Alpha-ketoglutarate to form

glutamate/-tic acid

Amine is used by carboxyphopshate to form carbamic acid

This is converted to carbamoyl phosphate

Carbamoyl Phosphate

Reacts w/ ornithine to form citrulline

Citrulline

Conjugated to aspartate to form argininosuccinate

Breaks down to form arginine and fumarate


15/23

Ornithine regenerated when producing urea by breaking arginine into

ornithine and urea

Above = urea cycle/ornithine cycle


16/23

Pentose Phosphate Pathways 11/6/2014 11:22:00 AM

PPP

Precursors for amino acid synthesis

Diverts a glycolysis intermediate

Produces starting material for nucleotide/histidine biosynthesis

(ribose-5-phosphate)o Starting material for aromatic amino acid (erythrose 4-

phosphate)

Major source of NADPH

Oxidative Phase

1ststep

o Start with Glucose-6-Phosphate

o G6P dehydrogenase (G6PD) catalyzes reaction

o NADPH generated

2ndstep

o Hydrolysis of the product from first step to form 6-

phosphogluconate

3rdstep

o Decarboxylation and oxidation bt 6-phosphogluconate

dehydrogenase

o Removes CO2 6-phosphogluconate and product becomes

Ribulose-5-phosphate

o

NADPH generatedNon-oxidative Phase

Mingle mangling produces Glyceraldehyde-3-phosphate (glycolysis)

o Erythrose4-phosphate (precursor for aromatic amino acid

synthesis)

o Ribose5-phosphate, xylulose-5-phosphate (precursor for

nucleotides)

o Fructose-6-phosphate (glycolysis)

o Sedoheptulose-7-phosphate

Ribulose-5-phosphate

Stays in PPP or isomerizes to ribose-5-phosphate for nucleotide

synthesis

Transketolase takes 2C from one xylulose-5-phosphate and adds it

to a ribose-5-P, making G3P (enters glycolysis or stays in PPP) and

Sedoheptulose7-P


17/23

Transaldolase

Takes 3C from sedoheptulose-7-P and generates fructose-6-P with

erythrose-4-P

Overall

2 NADPH generated/G6P Non-oxidative phase: other metabolic precursors generated

If no other processes were begun, 3G6P becomes 2 fructose 6 P

and 1 glyceraldehyde 3 P for glycolysis

Energy losing process

o More loss w/ more loss of carbons in R5P or E4P

-------------------------------Nucleotide Biosynthesis-------------------------

Nucleotide

Base + ribose + phosphate (s)

Nucleoside = nucleotide w/o phosphates

Cellular metabolism as energy source

Precursor to DNA/RNA etc

Consumption

At any time, the amount of nucleotides present is not enough to

perform duplication of genome in E. coli

Ribose-5-P with ATP is made to PRPP

Step highly regulated as PRPP cant be used for anything other than

nucleotide/histidine biosynthesisAfter PRPP generated

Salvage Pathway: 1 step to get adeneine and form nucleotide by

replacing PPi in the PRPP

Can also replace w/ UMP, CMP, TMP

Pyrimidine Biosynthesis

Begins w/ bicarbonate to carbomoyl phosphate process

o Bicarbonate acquires phosphate from ATP then loses it to take

NH2 then loses H on OH to get another Phosphate and form

carbamoyl phosphate

Cyclizes with aspartate to form orotate

Orotate reacts w/ PRPP to form orotidylate and eventually UMP

UMP becomes UDP and UTP (RNA)

UTP can be converted to CTP (replace O with NH2) and TTP can be

synthesized from UTP


18/23

Purine Synthesis

Gln used as NH2 donor to PRPP

Generates IMP which is converted to AMP or GMP (AMP needs GTP,

GMP needs ATP hehe)

Overall nucleotide biosynthesis = messay and consumes LOT of energySuppressed when

No Pi

Too much ADP


19/23

Regulation of Carbohydrate Metabolism11/6/2014 11:22:00 AM

Langerhans islets

Contain endocrine cells

Alpha and Beta Cells

o Alpha converts glucagon to make more glucose

o

Beta makes insulin to reduce blood glucoseHow B cells determine when to secrete insulin

Blood glucose high

GLUT2 in B cells imports glucose into cell

ATP in cell increases

ATP closes ATP gated K+ channel

Ca2+ import gets activated, stimulating insulin secretion

Weak affinity glucose transporter = key point

Km is similar to blood glucose concentration range

Transport range is dependent of glucose concentration in blood

How A cells determine when to secrete glucagon

Blood glucose low

GLUT2 in A cells import glucose into cells slowly

o ATP lowers

K+ pump gets inhibited

Ca2+ in cell stimulates glucagon secretion

Insulin

Peptide hormone from beta cells 2 chains w/ disulfide bonds w/ more carbs sometimes

signals liver and other organs to take up glucose by telling them

theres too much

Glucagon

Elevates blood glucose level

Peptide based hormone

Binds to liver cells and activates glycogen phosphorylase (no idea

how doe); breaks down glycogen to glucose-1-phosphate to form

glucose and release to blood

Adrenaline

Amino acid derived hormone from adrenal gland

Functions like glucagon but was easier to synthesize

Glycogen

Polymer of glucose


20/23

Gets a monosaccharide replaced with a phosphate group, releasing

that G1P

Phosphorylation: simple addition of phosphate

Phosphorylase: using phosphate as nucleophile to break something down

Glycogen Phosphorylase on Glycogen G1P becomes Glucose 1,6-bisphosphate and then glucose 6

phosphate

This can either go through gluconeogenesis or through glycolysis

Experiment

Grind up livers and apply epinephrine and glucagon

Bunch of different glycogen phosphorylase stuff

Found that cAMP turned glycogen phosphorylase into active R form

cAMP activated via GPCR

PKA

2 R and 2 C subunits

R units autoinhibit C subunits

cAMP binds to R (2 each cooperatively) and dissociate them from C

subunits

phosphorylates phosphorylase kinase which phosphorylates

glycogen phosphorylase to activate it

Also phosphorylates PFK2 to inhibit Fructose-2,6-bP synthesis

Suppresses glycolysis and increases gluconeogenesisInsulin signaling

Binds to Trk receptor on cell surface

Second part comes together to form dimer

Phosphorylation happens inside cell

IRS (insulin receptor substrate) becomes phosphorylated

Recruits PI3K

o Contains SH2 domain that binds to phosphorylated tyrosine

o Phosphorylates PIP2 to become PIP3

PIP3

o Recruits cytosolic proteins w/ PH domains and relocates them

to membrane

o Recruits PDK1 and AKT

Causes survival and ribosome synthesis and glucose

uptake


21/23

Point is to show translocation of glucose transporter from intracellular

vesicles to plasma membrane

Insulin goes upglucose sucked into the cell

Glycogen synthase also activated

Diabetes Type 1 = autoimmune

Type 2 = normal insulin level


22/23


23/23

Cholesterol = precursor for steroid hormone synthesis

Cholesterol derivative + UV light vitamin D vut? Just give me a ton of

cholesterol and a UV lamp. Ill be IRON MAN.

Cholesterol metabolism regulation

Too much cholesterol inhibits HMG CoA reductase = de novoshutdown

Transcriptional control

Hypercholersterolemia (HF)

o Oversynthesize cholesterol (hypercholesterol)

Normal people

o LDL internalized and delivered to ER

o SCAP-SREBP complex

SCAP binds to cholesterol and keeps it in ER when

abundant

SREB DNA-binding sequence cut when cholesterol levels

low

Goes to nucleus and activates HMG-CoA reductase

AMPK

PK complex that senses energy status

Binds AMP to gamma unit

Conformation changes and alpha subunit becomes phosphorylated

by upstream kinases Active and phosphorylates HMG CoA reductase and ACC

Low energyAMPK activatedphosphorylates ACC/HMG CoA

reductasefatty acid synthesis shut down along w/ cholesterol

synthesis

biochem exam 3

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