Biochemistry Unit 3 Material

gluconeogenesis

• synthesis of new glucose from non-carbohydrate precursors

  • precursors: pyruvate, lactate, amino acid, glycerol

• occurs primarily in the liver (90%) as well as the kidneys and small intestines

• important during long intervals between meals, overnight sleep, longer fasts, and rigorous exercise (when blood glucose levels decrease)

• 2 molecules of pyruvate are used to synthesize 1 molecule of glucose—bypasses the 3 irreversible steps of glycolysis (1,3,10)

• helped with enzymes:

  • pyruvate carboyxlase, PEP carboxykinase, fructose 1,6-bisphosphate, and glucose-6-phosphate

gluconeogenesis precursors

lactate, pyruvate, amino acids, glycerol

gluconeogenesis importance

• occurs because blood glucose levels are low or hypoglycemic

  • the brain completely relies and depends upon glucose as a primary source, so if glucose levels drop, the brain becomes affected

  • when fasting for a long period of time, the brain takes fuel from ketones (secondary source)

gluconeogenesis enzymes

1. pyruvate carboxylase

2. PEP carboxykinase

3. fructose 1,6-bisphosphatase

4. glucose-6-phosphatase

carboxylation of pyruvate

1. converts to PEP catalyzed by enzyme pyruvate carboxylase

• pyruvate carboxylase is present in mitochondria

• pyruvate must be transported from cytoplasm to mitochondria via mitochondrial pyruvate carrier

• within the mitochondria, pyruvate is converted to oxaloacetate by using the energy 2 molecules of ATP

• the formation of PEP from pyruvate requires pyruvate carboxylase + phosphoenolpyruvate carboxylase

carboxylase

a ligase that catalyzes the formation of C-C bonds, typically involving the addition of a carboxyl group (CO2) to a molecule

oxaloacetate converted to malate

2. 
   

• oxaloacetate has to be transported out of the mitochondria

• mitochondria has a special gate to transfer malate with the help of malate-aspartate shuttle

• oxaloacetate is converted to malate with the help of malate dehydrogenase and 2 molecules of NADH are oxidized to NAD+

• malate is now transported out in the cytoplasm to be converted to phophoenolpyruvate

malate to PEP

3. 
   

• 2 malate in the cytosol are then converted back into oxaloacetate with the help of the cytosolic form of malate dehydrogenase enzyme

  • NAD+is reduced to 2 NADH

  • NADH enters ETC to synthesize ATP

• oxaloacetate is converted to PEP with help of PEP carboxykinase enzyme

• requires 2 GTP and releases CO2

conversion of F-1,6-BP

• PEP, through a series of reactions, is then converted into fructose 1,6 bisphosphate, 2 molecules of ATP are being used

• the fructose-1-6-bisphosphate is converted to F6P using fructose 1,6, bisphosphatase (allosteric enzyme)

• irreversible step

generation of glucose from G6P

• F6P → G6P → glucose

  • glucose-6-phosphatase (enzyme that releases free phosphate)

• in most tissues, one G6P is generated and gluconeogenesis stops

  • it does not release glucose until its required (only when blood glucose is low)

  • would then by generated into glycogen (to store glucose until needed)

  • glucose is not generated in the cytosol

• G6P must be transported into the lumen of smooth ER, then can be hydrolyzed into glucose and inorganic phosphate (by membrane bound G6P enzyme)

  • then the glucose will be shuttled back into the cytosol with transporters 2 and 3

• free glucose level is highly maintained by liver and kidney

gluconeogenesis energy

• 4 ATP, 2 GTP, and 2 NADH are used to breakdown 2 pyruvate

  • net output: -6 ATP

triglyceride to glucose

• triglycerides are broken down into glycerol and fatty acids (lipolysis)

  • releases energy for the body to use

• glycerol is transported into liver cells

  • glycerol → glycerol-3-phosphate (via enzyme glycerol kinase)

• glycerol-3-phosphate enters the glycolytic pathway at the step of DHAP by the enzyme glycerol-3-phosphate dehydrogenase by glycerol-3-phosphate shuttle

• DHAP is converted to glucose through a series of steps in gluconeogenesis

glucogenic amino acids

• an amino acid that breaks down into pyruvate or a citric acid cycle (TCA) intermediate, which can be used to synthesize glucose via gluconeogenesis in the liver. [1, 2]

  • they are deaminated (removal of amino group) producing pyruvate or other intermediates

  • intermediates enter TCA or glycolysis where they are converted to glucose

  • glucose is then released into bloodstream

low blood glucose

• hormonal regulation of gluconeogenesis

  • level of glucagon hormone rises, which activates adenylate cyclase enzyme

    • catalyzes the conversion of ATP→ cAMP

    • cAMP activates protein kinase A enzyme, which adds a phosphate group to the pyruvate kinase enzyme

    • phosphorylation renders pyruvate kinase enzyme inactive → no PEP to pyruvate

    • pyruvate converts to oxaloacetate and PEP, followed by a series of reactions by gluconeogenesis

high blood glucose

• hormonal regulation of gluconeogenesis

  • increases insulin level

  • activates the protein phosphatase enzyme, which removes the phosphate group from pyruvate kinase

  • this activates it, catalyzing the conversion of PEP to pyruvate, which can no longer be converted to oxaloacetate

    • activates the glycolysis pathway

breakdown of glucose

• breakdown of carbs → glucose

  • once glucose is in the cell, hexokinase attaches 6th phosphate → G6P

    • from there, glycolysis can occur

  • If ATP is not needed, glucose can be used to make other useful products via the pentose phosphate pathway

pentose phosphate pathway

• named for its products

  • pentose- 5C (ribose)

    • used to form nucleotides (building blocks for RNA/DNA)

  • phosphate- NADPH

    • rich in electrons, used in anabolic pathways. used in various pathways such as in the synthesis of fatty acids

• alternative pathway when cells need ribose + NADPH

• has no clear start/end

• happens exclusively in the cytoplasm

• can be divided into irreversible oxidative phase and reversible nonoxidative phase

• role is serve as a cellular interchange for materials

oxidative phase

• first phase of PPP

1. glucose-6-phosphate dehydrogenase snatches H from G6P and gives it to NADP+ → making NADPH + 6-phopho-gluconate [RATE DETERMINING STEP]

2. 6-phosphogluconate dehydrogenase steals electron from 6-phosphogluconate and gives it to NADP+ → making another NADPH + ribulose-5-phosphate + CO2

• end of phase = 2NADPH produced from 1 glucose

• irreversible due to CO2 being released as a gas (hard to retrieve) and the cells wanting NADPH to be present (favorable)

nonoxidative phase

• 2nd phase of PPP

• reversible

• generates products due to cell needs

1. ribulose 5 phosphate gets converted to ribose-5-phosphate by an isomerase enzyme

• ribose-5-phosphate is important in nucloetide synthesis, the

2. alternatively, ribulose-5-phosphate can be converted into 5C sugar, xylulose-5-phosphate by an epimerase enzyme [happens when the cell does not want to make RNA/DNA]

   1. transketolase removes 2C from xylulose 5 phosphate and adds it to 5C ribose-5-phosphate → 7C sedoheptulose-7-phosphate and 3C G3P

   2. transaldolase removes 3C from sedoheptulose-7-phosphate turning it into 4C erythose-4-phsohpate

      1. the 3C go to G3P making F6P

• both G3P and F6P are intermediates in glycolysis [so the cell can divert if needed]

epimerase

• type of isomerase enzyme

• ex: used in PPP (nonoxidative phase)

transketolase

• type of transferase; the reversible transfer of a keto group from ketose to an aldolase

• ex: used in PPP (nonoxidative phase)

hexose monophosphate shunt

• if the cell is in the middle of glycolysis, but needs nucleotides instead, transketolase + transaldolase can convert G3P and F6P back to phosphate → ribose-5-phosphate

• pathway allows for carbon atoms from G6P to take a detour before they proceed down the glycolytic pathway

• PPP allows cells to use whatever sugar phosphate molecules they ned to make other compounds for cells

amino acid functions

• building blocks of proteins [10-15% of energy]

• synthesis of nucleic acids

• synthesis of neurotransmitters

• part of prosthetic group as porphyrins (ex: histadine)

fed state

• high insulin, low glucagon

  • protein is broken down into amino acids in the intestine and transported via the blood to the liver

  • in the liver, the amino acids are used for protein synthesis or converted into glucose and fatty acids

  • glucose is stored as glycogen in liver

  • fatty acids are stored as triglycerides in adipose tissue

  • glucogenic AA

  • ketogenic AA

glucogenic amino acids

• amino acids that can be converted into glucose through gluconeogenesis in the liver

ketogenic amino acids

• amino acids that are directly degraded in acetyl-CoA or acetacetyl-CoA rather than glucose, making precursors for ketone bodies and fatty acids

fasted state

• low insulin, high glucagon

  • fatty acids are released from adipose tissue and sent to the liver, where they are oxidized and ATP fuels the synthesis of glucose

  • amino acids are released from muscles to the liver and enter diverse arrays of metabolic pathways

  • glucogeneic amino acids are released, providing precursors for gluconeogenesis and helping produce glucose

essential amino acids

• 9 amino acids

• body cannot create on own, must be obtained through foods

  • phenylalanine

  • valine

  • tryptophan

  • threonine

  • isoleucine

  • methionine

  • histidine

  • leucine

  • lysine

• instead of body storing these, the body uses them regularly to make new proteins

nonessential amino acids

• 11 amino acids

• synthesized in body

• essential for protein synthesis and do not need to be included in diet

  • alanine

  • aspartate

  • glycine

  • serine

  • cysteine

  • glutamic

  • ornithine

  • proline

  • tyrosine

biosynthesis of amino acids

• carbon skeleton comes from intermediates of glycolysis, citric acid cycle, or PPP

• no single unifying pathway for synthesizing all amino acids

transamination reactions

• reactions that contribute to formation of amino acids

• transfers an amino group from 1 molecule to another

  • converts a keto acid into an amino acid

• usually glutamate acts as a donor/acceptor

• aspartate, alanine, glutamate are formed by addition of an amino group to an alpha-keto acid

oxidative deamination

• glutamate is broken down by removing an amino group to produce ammonia (NH3) and a-ketglutarate

• requires NADP+ being reduced to NADPH by glutamate dehydrogenase; in process NH4 is lost

• ammonium is converted to urea via the urea cycle → later excreted by the kidneys

glutamate → a-ketoglutarate

amidation reactions

• reactions that contribute to amino acid formation

  • directly adds an amide group to a molecule

  • glutamate → glutamine (via glutamine synthetase)

  • aspartate + NH3 + ATP → asparagine (via asparagine synthetase) + ADP + P

    • forms an adenylated intermediate

one carbon transfer reactions

• 3 phosphoglycerate (glycolysis) is the precursor of serine, cysteine, and glycine

  • serine + tetrahydrofolate → glycine

  • requires tetrahydrofolate (THF) is a derivative of vitamin B9 (folate)

biosynthesis of aromatic amino acids in plants

• plants, bacteria, fungi

  • can produce tryptophan, tyrosine, and phenylalanine using Shikimate-Chorismate pathway

    • process begins with condensation of PEP (Glyc) and erythrose-4-phosphate (PPP) to form Chorismate

    • chorismate → anthranilate (condenses with PRPP) → tryptophan

    • chorismate → prephenate → tyrosine/phenylalanine

biosynthesis of aromatic amino acids in humans

• phenylalanine (essential a.a.) is converted to tyrosine with phenylalanine hydroxylase

nitrogen fixation

• few plants can partner with nitrogen fixing bacteria (symbiosis) to capture nitrogen

• rhizobium bacteria invade the roots of leguminous plants and form root nodules to fix nitrogen

• diazotrophic (nitrogen fixing) microorgansims fix ~60% of Earth’s nitrogen

nitrogenase complex

• located inside nitrogen fixing bacteria (cytoplasm), carries out nitrogen fixation process, converting atmospheric nitrogen into ammonia

• 2 components:

  • reductase: funnels electrons from Fe-S protein to nitrogenase

  • nitrogenase: catalyzes the reduction of nitrogen to ammonia

    • inactivated by oxygen and need protection (leghemoglobin)

leghemoglobin

• homolog of hemoglobin

• binds with oxygen in nodules to lower free oxygen concentration in bacteria and allow nitrogenase to make NH3 (ammonia) or NH4 (ammonium) in water

• resembles myogblobin

• affinity of oxygen is very high, 10x that of hemoglobin

impossible foods

• uses soy leghemoglobin in their impossible burger to give it meaty flavor and texture

non-legumes amino acid synthesis

• cannot take up atmospheric nitrogen directly because it exists in a gaseous form with strong triple bonds between nitrogen atoms

  • instead, they absorb N in the form of nitrates/nitrites from the soil

• nitrate is in the soil and taken up by plant roots and reduced to nitrite (nitrate reductase)

• nitrite is stored in vacuoles of root cells or transported to the leaves

  • nitrite reductase catalyzes reduction of nitrite to ammonium

• next step is assimilating N into biomolecules is the entry of NH4 into amino acids

  • glutamate and glutamine play pivotal roles in acting as nitrogen donors

nucleotides

• nitrogenase base + pentose sugar + phosphate

• Purine: N9 connects to C1 of sugar by glycosidic linkage (A and G)

• Pyrimidine: N1 connects to C1 of sugar by glycosidic linkage (C,U and T)

• both bases are flat in the ring plane

cytosine

2-oxy-4-aminopyrimidine

• can be converted to uracil by the deamination process, releasing NH4+

uracil

2-4-dioxypyrimidine

thymine

2-4-dioxy,5-methylpyrimidine

pyrimidine structure

• nitrogenous base, 6 membered ring, single ring structure

• presence at N at position 1 and 3

• Used for DNA synthesis

• Requires:

  • glutamine, aspartate, tetrahydrofolate (Vitamin B9)

• occurs in cytoplasm

• only anti form is allowed (oxygen points away from sugar)

• crucial for genetic replication, transcription, and cell division

synthesis of PRPP

ribose-t-phosphate is converted to phosphoribosyl-a-pyrophosphate (PRPP)

• enzyme: ribose phosphate pyrophosphokinase

  • ATP → AMP (takes 2 phosphates from ATP)

• end produce is used for amino acid biosynthesis (purine/pyrimidine)

pyrimidine synthesis

• starts with PRPP

• key components: glutamine, bicarbonate, ATP, H2O lead to the formation of carbamoyl phosphate (CPS-2)

  • CPS-2 catalyzes the rate determining step, converting glutamine, CO2, and ATP into carbamoyl phosphate (critical regulatory step and is subject to feedback inhibition by UTP)

• conversion of orotic acid: series of reactions, carbamoyl phosphate reacts with aspartate to from carbamoyl asparate, which is then cyclized to produce dihydroororate.

  • is subsequently oxidized by the enzyme dihydrooroate (DHODH) to form orotic acid

• formation of UMP: orotic acid undergoes a phosphoribosylation step to from OMP.

  • OMP dicarboxylase then catalyzes the removal of a carboxyl gruop to make UMP *first nucletoide produced in pathway

  • once UMP is formed, it can be phosphorylated to form UDP and UTP, which can be further converted into other pyrimidine nucleotides, including CTP and, in DNA synthesis, dTTP

ribonucleotide reductase

• converts the ribose to deoxyribose for converting UDP to dUDP

  • followed by kinase that adds to the phosphate to make dUTP.

• dUTP is converted to dUMP by dUTPase and dUMP synthesizes dTMP with thymidylate synthase and tetrahydrofolate.

pyrimidine ring sources

• Aspartate = N1, C4, C5, C6

• Glutamine = N3

• CO2= C1

purine structure

• 6 membered ring fused to a 5 membered ring

• Glutamine and Adenine

• form the energy carrier molecules: ATP and GTP

• requires glutamine, glycine, aspartate, and tetrahydrofolate (vitamin B9)

• can be synthesized using a de noro or salvage pathway

• synthesis occurs in the cytoplasm

adenine

6-aminopurine

guanine

2-amino-6-oxypurine

purine synthesis

• PRPP is converted to IMP with the help of glycine, tetrahydrofolate, glutamine, ATP, CO2, aspartate and ATP, and tetrahydrofolate molecule (10 steps)

  • IMP is the precursor for purine nucleotide AMP and GMP

  • further phosphate additions to generate diphosphate and triphosphate nucleosides may follow the completion of monophosphate synthesis (carried out by kinases)

• both ATP and GTP are then converted to dATP and dGTP with ribonucletoide reductase that will be used for DNA synthesis

ATP synthesis

• IMP is first converted to adenylosuccinate with the help of adenosuccinate synthetase

• the adenylosuccinate is then converted to AMP with the release of fumarate.

• AMP is converted to ADP with AMP kinase and further to ATP with ADP kinase

• with ribonucleotide reductase, AMP is converted to dADP and then to dAMP

GTP synthesis

• IMP forms XMP with enzyme IMP dehydrogenase

• XMP converts to GMP using GMP synthase

• with help of guanylate kinase, GMP is converted to GDP

• with the help of GDP kinase, GDP is converetd to GTP

purine ring sources

• aspartate: N1

• THFA: C2 and C8

• Glutamine: N3 and N9

• Glycine: C4, C5, and C7

• CO2: C6