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Biochemistry > Biochem 4 > Flashcards

Biochem 4 Flashcards

1
Q

overview of metabolism

A
  • different organisms use different strats for capturing free energy from their environment and can be classified by their requirement for O2
  • mammal nutrition involves intake of macronutrients (proteins, carbs, lipids) and micronutrients (vitamins and minerals)
  • metabolic pathway is a series of enzyme-catalyzed rxns usually located in specific part of cell
  • flux of material through a metabolic pathway varies with the activities of the enzymes that catalyze irreversible (or quasi-irreversible) rxns
  • flux controlling enzymes are regulated by allosteric mechanisms, covalent modification, substrate cycling, and change in gene expression
  • regulates rate of enzyme synthesis, rate of enzyme degradation, modification of enzyme affinity, catalytic activity by small molecules that bind allosterically, interactions with enzymes with each other, subunit interaction
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2
Q

the more enzyme

A
  • the faster the rxn

- regulatory strategy for metabolic pathways

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3
Q

high energy phosphates

A
  • energy storage molecules
  • ATP
  • phosphodiester linkages
  • phosphoanhydride
  • one of two major pathway for breakdown and synthesis of protein, lipids and carbs
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4
Q

reducing equivalents

A
  • energy storage molecules
  • NAD
  • NADP
  • when they are reduced they are sources of energy
  • one of two major pathway for breakdown and synthesis of protein, lipids and carbs
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5
Q

ATP

A
  • alpha phosphate is linked to the sugar of adenosine through phosphoester bond
  • adenine -> the base
  • adenosine -> the base with the sugar
  • beta and gamma phosphates are linked with phosophoanhydride bonds (very reactive)
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6
Q

catabolism

A

-breakdown

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7
Q

metabolism for carbohydrates: How do we get the reducing equivalents?

A
  • How do we get the reducing equivalents NAD and NADP and FAD and FMN as well as the high energy phosphate ATP out of catabolism of carbohydrates
  • these pathways are not just for breakdown
  • there are points of escape that permit us to use these pathways for biosynthetic purposes for other metabolites
  • use catabolic pathways to make ATP, reducing equivalents, FAD, FADH2 -> but also for biosynthesis
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8
Q

oxidation

A
  • loss of e-
  • most oxidized form of C is CO2
  • on its way to getting oxidized to CO2 it gets a double bond, an alcohol, a carbonyl, carboxylic acid intermediates
  • important for carbohydrate metabolism
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9
Q

reduction

A

gain of e-

-most reduced form of C is methane

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10
Q

carbohydrate catabolism

A
  • initial metabolic intermediates that are broken down in carbohydrate catabolism are typically reduced forms that have few carbonyls or alcohols -> eventually oxidized to carbonyls and carboxylic acids -> finally CO2
  • metabolites are water soluble -> bounce around easily in the cytosol
  • needs to be regulated to package the rxns in the cytosol and separate from the cytosol (organelles like mitochondria)
  • initial breakdown of carbohydrates like glucose takes place in cytosol
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11
Q

compartmentalization

A
  • important regulatory strategy for metabolism
  • capturing energy for carbohydrate metabolism takes place in mitochondria
  • mitochondria is a catabolic organ
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12
Q

anabolic or synthetic rxns

A

-occur in the cytosol

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13
Q

cytosol

A

-how does the cytosol know whether to use the rxn pathways to breakdown or synthesize ?

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14
Q

heme

A

-hemoglobin has a need for heme to hold to iron in its reduced form
Synthetic pathway:
-1st step- rate limiting step of the heme biosynthetic pathway
-that enzyme is regulated -> if its not being used it is rapidly broken down -> enzyme is constantly being synthesized and broken down
-extreme regulation for heme synthesis

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15
Q

allosteric regulation

A
  • regulates metabolism

- small molecules, coenzymes, or proteins that allosterically modify activity of enzymes

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16
Q

covalent modifications of enzymes

A

-phosphorylate or dephosphorylate enzymes that critical to pathways

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17
Q

rate of rxn depends on concentration of substrates

A
  • not always the best method
  • when substrate concentration exceeds the dissociation constant for the substrate from the enzyme then the rxn becomes 0th order with respect to substrate concentration
  • rate is more sensitive to concentration at low concentrations
  • chemical kinetics: frequency of substrate meeting the enzyme matters
  • rate becomes insensitive at high substrate concentrations
  • enzyme is nearly saturated with substrate
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18
Q

rate of rxn depends on concentration of substrates: blood glucose example: hexokinase

A
  • 2 enzymes involved in trapping glucose in cells
  • glucokinase- find in liver cells
  • hexokinase- found everywhere else
  • glucose gets phosphorylated and becomes glucose 6 phosphate
  • Km for hexokinase is on the order of 120th of the blood glucose concentration -> as long as glucose can enter the cell through transporters -> rate of conversion of glucose to glucose 6 phosphate is independent of the blood glucose concentration bc physiological normal blood glucose concentrations are 20x the Km of hexokinase
  • rate of conversion of glucose to glucose 6 phosphate is determined by the levels of hexokinase
  • *function of hexokinase is not simply to phosphorylate glucose -> it is to trap glucose inside cells (due to a neg charge)
  • it will trap glucose at a rate that is independent of blood glucose concentration
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19
Q

rate of rxn depends on concentration of substrates: blood glucose example: glucokinase

A
  • 2 enzymes involved in trapping glucose in cells
  • glucokinase- find in liver cells
  • hexokinase- found everywhere else
  • liver is heavy lifting organ when it comes to regulation of metabolism
  • *glucokinase is capable of regulating the substrate level of blood glucose level bc it works more rapidly when blood glucose exceed Km and less rapidly when they are below Km
  • glucokinase Km is twice the glucose blood concentration -> glucokinase having glucose bound is very dependent on the blood glucose concentration bc the Km is right around the Km for blood glucose
  • glucokinase will phosphorylate more rapidly if the blood concentration goes up and less rapidly if the blood glucose goes down
  • liver is sacrificial -> even if it might need glucose for its own metabolism it wont extract it out of the blood if blood glucose is low but it will if blood glucose is high and store it as a polymer (glycogen)
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20
Q

rates of a biochemical rxn

A
  • depends on:
  • concentration of reactant vs product
  • activity of the catalyst (concentration of the enzyme -> rate of translation versus rate of degradation, intrinsic activity of the enzyme -> could depend on substrate, effectors, or phosphorylation state)
  • concentration of effectors (allosteric regulators, competing substrates, pH, ionic environment)
  • temperature
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21
Q

metabolic pathways

A
  • many metabolic rxns are catalyzed by enzymes that are functioning more or less at equilibrium -> but there are a couple of steps that are virtually irreversible (ΔG«0) -> committed steps
  • committed steps determine the directionality of the pathway
  • steps that are highly regulated
  • catabolism and anabolism are controlled differentially -> keep products in different compartments in the cell, allows both to be virtually irreversible, independent control of each (can occur at same time)
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22
Q

reactions far from equilibrium are common points of regulation

A
  • when a rxn is near equilibrium typically that rxn is unregulated
  • enzyme involved is function with no significant modulation either through allosteric mechanisms or substrate concentrations
  • these enzymes are simply functioning as good catalysts
  • if rxn is function far from equilibrium -> regulatory mechanisms kick in
  • to maintain steady state, all enzymes operate at the same rate
  • good catalysts (function near equilibrium)- aldolase, triose phosphate isomerase, phosphoglycerate mutase, enolase, pyruvate kinase, phosphoglucose ismoerase, glucose-6-phosphate, glyceraldehyde-3-phosphate dehydrogenase + phosphoglycerate
  • regulatory (function far from equilibrium)- hexokinase, PFK-1, pyruvate kinase, pyruvate carboxylase + PEP
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23
Q

vitamins treat diseases- B vitamins

A
  • small molecules that are cofactors for the enzymatic rxns -> vitamins (catabolic or anabolic)
  • nicotinamide (niacinamide) -> B3 -> important for reduction rxn -> NAD and NADP
  • nicotinic acid (niacin)
  • some can be made within human body (low concentration)
  • thiamine- B1 -> part of the cofactor thiamine pyrophosphate -> important for carbonyl carbon rxns
  • redox compounds- FAD and FMN contain the riboflavin (B2)
  • pantothenic acid- B5 -> transport cofactor -> coenzyme A -> moves metabolites between the cytosol and mitochondria
  • B vitamins are water soluble
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24
Q

fat soluble vitamins

A
  • lipid rxns
  • involved in rxns hat involve fat soluble membranes
  • vitamin A- Vision -> night blindness
  • vitamin D- Ca2+ absorption -> rickets
  • vitamin E- antioxidant
  • vitamin K- blood clotting -> hemorrhage
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25
Q

ATP, ADP, AMP, and Adenosine

A
  • anhydride linkages- beta and gamma phosphates -> capture energy
  • phosphodiester bond- alpha phosphate and adenosine
  • free energy of hydrolysis of a # of high or low energy phosphates varies
  • enol phosphate (phosphoenolpyruvate) -> very high free energy of hydrolysis
  • nitrogen phosphate (phosphocreatine) -> important for energy reserve in muscles
  • phosphoesters (glucose-1,6,3-phosphates) -> low energy phosphates
  • thioester- high energy
  • aminophosphate- high energy
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26
Q

endergonic rxns coupled to ATP hydrolysis

A
  • drive rxns involving placement of phosphate onto various metabolites
  • phosphorylation of glucose to glucose-6-phosphate by hexokinase is a way to trap it inside cells -> energy consuming -> therefore you need to couple ATP hydrolysis
  • ATP donates the phosphate and the conversion to ADP is the driving force -> coupled rxns
  • phosphate transfer rxn
  • if we break the phosphate transfer rxn into 2 half rxns -> it is apparent the exergonic (energy release) associated with hydrolysis of ATP is the driving force to achieve the direction of the endergonic phosphorylation of glucose
  • ADP can be phosphorylated by phosphoenolpyruvate by using the enolphosphate as the high energy exergonic reactant and the ADP as the lower energy reactant that can be phosphorylated
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27
Q

redox potential

A

-redox rxn involving FAD and FMN and NAD and NADP are also ways of trapping energy and determining rxn direction

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28
Q

driving rxn direction

A
  • phosphorylation

- redox reactions

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29
Q

use of amino acids as fuel varies greatly by organism

A
  • during oxidation amino acids release energy
  • 90% of energy needs of carnivores can be met by amino acids after a meal
  • microorganisms scavenge amino acids from their environment for fuel when needed
  • only a small fraction of energy needs of herbivores are met by amino acids
  • plants do not use amino acids as a fuel source but can degrade amino acids to form other metabolites (use light, CO2 to make glucose)
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30
Q

metabolic circumstances of amino acid oxidation

A
  • when we eat food proteins are broken down into amino acids
  • amino acids are taken up where needed (organs) and used
  • dietary amino acids that exceed bodys protein synthesis needs
  • leftover amino acids from normal protein turnover (proteolysis and regeneration of proteins) -> is broken down (catabolism), recycled and excreted out
  • we cannot store excess protein -> must be excreted
  • proteins in the body can be broken down to supply amino acids for energy when carbohydrates are scarce (starvation, diabetes mellitus)
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31
Q

dietary protein is enzymatically degraded through the digestive tract

A
  • we get protein from dietary foods
  • partially broken down in mouth
  • proteins get broken down in stomach (acidic) through enzymes (pepsin)
  • pepsin cuts protein into peptides in the stomach
  • in the small intestine (more neutral) pancreatic enzymes break down
  • trypsin and chymotrypsin cut proteins and larger peptides into smaller peptides in the small intestine
  • aminopeptidase (cuts form N terminal) and carboxypeptidase (cuts from C terminal) A and B degrade peptides into amino acids in the small intestine
  • protein -> peptide -> amino acids
  • villi absorbs amino acids -> circulatory system -> tissues
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32
Q

protases

A
  • enzymes that break down peptide bones through hydrolysis reactions (uses water)
  • degrade proteins
  • usually occurs in the lysosomes (mostly non-selective) or in the cytosol by the ubiquitin-proteasome pathway inside the cell (selective)
  • endopeptidases- cut in the middle
  • exopeptidases- cut at the end
  • aminopeptidase (cuts form N terminal)
  • carboxypeptidase (cuts from C terminal)
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33
Q

intracellular protein degradation: lysozyme

A
  • contain large # of different enzymes
  • can break down a variety of biomolecules (peptides, nucelic acids, lipids and carbohydrates)
  • digest food, organelles and even cells
  • things are picked up by phagocytosis
  • very acidic interior (pH 4.5-5)
  • pumps protons in via proton pumps and ion channels
  • fuse with other vesicles, organelles or structures (phagocytosis)
  • autophagy- autophagosome fuses with an organelle and then fuses with lysosome for breakdown
  • lysosome is encapsulated so it doesnt break down everything
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34
Q

proteins are selectively degraded by ubiquitin-proteasome pathway

A
  • cells must constantly recycle proteins/amino acids
  • unwanted proteins are targeted for breakdown
  • polyubiquitin (>4 ubiquitin chains) is the signal for protein degradation
  • tags proteins that needs to be degraded
  • proteasome is doing the breakdown
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35
Q

ubiquitin tag

A
  • ligases help binding or associating ubiquitin to the protein
  • ubiquitin is conjugated to proteins with the help of 3 types of enzymes
  • Ubiquitin-activating enzyme (E1)- ATP dependent rxn
  • ubiquitin adds to E1 through a cysteine
  • E1 binds to ubiquitin using hydrolysis
  • Ubiquitin-conjugating enzyme (E2)- ubiquitin transferred to E2 cysteine
  • E2 takes to ubiquitin from E1 and associates with it
  • ubiquitin protein ligase (E3)- E3 recruits target protein
  • responsible for binding target protein
  • E3 has 2 pockets (one for the protein and the other is E2 containing the ubiquitin molecules) -> takes off the ubiquitin from E2
  • process repeats until there at more than 4 ubiquitin’s tagging the protein
  • deubiquitinating enzymes (DUBs) can reverse the process -> takes off polyubiquitin chain form the protein
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36
Q

specificity of ubiquitin degradation

A
  • in humans:
  • there is 1 E1
  • there are 40 E2’s
  • there are > 600 E3’s (four major classes)
  • E3’s only recognize a certain subset of proteins
  • post-translational modifications can alter specificity
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37
Q

proteasome

A
  • very large multi-protein complex- function is to degrade polyubiquitinated proteins
  • composed of 4 stacked rings (alpha/beta/beta/alpha) -> symmetric
  • only the beta subunits (central enzymes) have proteolytic activity
  • alpha subunit must change to an open conformation (ATP-dependent) and unwind proteins (ATP-dependent) to pass them into the proteasome core for degradation (beta)
  • alpha subunit is an ATPase -> uses ATP hydrolysis to unwind protein
  • ubiquitin tag is cleaved off and the proteasome starts breakdown
  • 26S proteasome is made up of a hollow core (20S proteasome) and 19S cap
  • 19S cap recognizes polyubiquitinated proteins, unfolds them and passeS them into the 20S proteasome (not proteolytic)
  • 20S has large chambers that allow access to the proteases that hydrolyze protein chains
  • our cells can build specialized proteasomes by installing different caps
  • immunoproteasome generated during immune response can generate peptides that are used for major histocompatibility complex
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38
Q

overview of amino acid catabolism

A
  • proteins are made up of polypeptide chains made up amino acids
  • polypeptide chains are broken down into amino acids
  • amino acids are further metabolized:
  • Transamination- amino group gets transferred (R1) to another amino acid
  • oxidative deamination- broken down into carbon skeleton -> amino group is released as ammonia -> 2 products: carbon skeleton and ammonia
  • carbon backbone is fed into metabolism (glycolysis, citric acid cycle) -> make carbon molecules or glucose
  • ammonia goes into urea cycle or biosynthesis or amino acids
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39
Q

excretory forms of nitrogen

A
  • plants conserve almost all the nitrogen
  • ammonotelic- aquatic vertebrates release ammonia (toxic) to their environment (passive diffusion from epithelial cells and active transport via gills)
  • ureotelic- many terrestrial vertebrates and sharks excrete nitrogen in the form of urea (humans)
  • urea is far less toxic than ammonia
  • urea has very high solubility
  • uricotelic- some animals such as birds and reptiles excrete nitrogen as uric acid -> insoluble
  • excretion of uric acid as paste allows animal to conserve water
  • humans and great apes excrete both urea (from amino acids) and uric acid (from purines)
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40
Q

excretion of nitrogen: step 1: removal of amino group

A
  • release of free ammonia is toxic
  • ammonia is captured by a series of transaminations
  • transaminations allow transfer of an amine to a common metabolite (alpha-ketoglutarate) and generate a traffickable amino acid (glutamate)
  • alpha-ketoglutarate accept the amine group from the amino acid being broken down -> converted to glutamate
  • carbon skeleton gets converted into the alpha-ketoglutarate
  • transfer of amino group is done by the enzyme amino transferase
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41
Q

enzymatic transamination ex. 1

A
  • amino acid and keto acids are interconverted by transamination
  • amino acid in the presence of the alpha-ketoglutarate
  • amino acid loses its amine group and becomes a alpha-keto acid
  • alpha-ketoglutarate accepts the amine group and becomes glutamate (amino acid)
  • transfer of amino group is done by the enzyme amino transferase
  • bi-directional -> glutamate can transfer its amine group to another alpha-keto acid and convert it back into an amino acid (glutamate turns back into alpha-ketoglutarate)
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42
Q

ketones

A
  • ketone bodies
  • acetoacetate
  • beta-hydroxybutyrate
  • also acetone keto acids
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43
Q

enzymatic transamination ex. 2

A
  • oxaloacetate- keto acid
  • oxaloacetate accepts the amine group from glutamate
  • oxaloacetate forms aspartate
  • glutamate becomes alpha ketoglutarate
  • transfer of amino group is done by the enzyme amino transferase
  • bi-directional
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44
Q

pyridoxal phosphate (PLP)

A
  • pyridoxal-5’-phosphate (PLP)
  • coenzyme helps enzyme (amino transferase) do its job
  • derived from pyridoxine (vitamin B6) -> becomes PLP when phosphorylated
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45
Q

transamination rxn using pyridoxal phosphate (PLP)

A
  • aldehyde form of PLP can react reversibly with amino groups -> becomes a pyridoxamine phosphate
  • pyridoxamine phosphate accepts another amino group and transfers it onto another keto (intermediate)
  • aminated pyridoxamine phosphate can react reversibly with carbonyl
  • transaminase enzyme
  • intermediate, enzyme bound carrier of amino acids- carrier of the bound amino group
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46
Q

pyridoxal phosphate is covalently linked to the enzyme is the resting enzyme

A
  • by an internal aldimine
  • pyridoxal phosphate is associated with transaminase enzyme (aminotransferase) through an internal aldimine
  • lysine as a amino molecule that attaches to the pyridoxal phosphate -> forms shift base conjugate of the enzyme -> active site lysine molecule
  • the linkage is made via a nucleophilic attack of the amino group of an active site lysine
  • coenzyme associated with enzyme
  • lys is associated with PLP through a shift base
  • an amino acid that needs to transfer its amino group forms a bond with PLP within the lysine active site
  • water comes in and converts amino acid into keto acid
  • amine group is transferred to pyridoxal phosphate -> pyridoxamine phosphate
  • alpha-ketoglutarate comes in an forms a bond with amine group -> takes the amine group from the pyridoxamine phosphate -> becomes amino acid -> glutamate
  • pyridoxamine phosphate finds lysine
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47
Q

PLP based deamination of amino acids

A
  • amino group is removed from the amino acid and passed onto the keto acid
  • keto acid is converted to amino acid
  • amino acid is converted to keto acid
  • pyridoxal phosphate (internal aldimine form) associated with transaminase (aminotransferase) enzyme
  • amino acid comes in and associates with the pyridoxal phosphate in the amine form
  • amine form transfers its amino group onto the pyridoxal phosphate
  • amino acid gets converted to keto form
  • amine group on original amino acid gets transferred to the pyridoxamine phosphate
  • facilitated by the cofactor
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48
Q

PLP also catalyzes racemization of amino acids

A
  • most of the amino acids is L-amino acid
  • L-amino acid gets converted to D-amino acid by racemization
  • facilitated by PLP cofactor
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49
Q

PLP also catalyzes decarboxylation of amino acids

A
  • amino acid loses its carbonyl acid group
  • CO2 is released
  • pyridoxal phosphate goes back pyridoxal phosphate enzyme conjugated pyridoxal phosphate form -> releases an amine
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50
Q

PLP based transamination is “ping pong bi bi” rxn

A
  • strictly sequential process
  • 2 substrates IN
  • 2 substrates OUT
  • amino acid IN (attaches to pyridoxal phosphate)
  • alpha keto acid OUT (alpha-keto acid)
  • alpha-keto acid IN (alpha-ketoglutarate)
  • alpha-amino acid OUT (glutamate)
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51
Q

summary of enzymatic transamination

A
  • catalyzed by aminotransferases
  • uses pyridoxal phosphate cofactor
  • typically, alpha-ketoglutarate accepts amino groups
  • transfer 1 amine to alpha-ketoglutarate results in synthesis of glutamate (transamination)
  • transfer of a second amine results in synthesis of glutamine (glutamine synthetase)
  • L-glutamine acts as a temporary storage of nitrogen
  • L-glutamine can donate the amino group when needed from amino acid biosynthesis
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52
Q

ammonia is safely transported in the bloodstream as glutamine

A
  • ammonia is toxic and must be transported in safe forms throughout the body from tissue to tissue
  • excess ammonia in the body/blood is added to glutamate and converted to glutamine using glutamine synthetase
  • L-glutamate accepts ammonia -> glutamine (uses ATP)
  • catalyzed by glutamine synthetase
  • glutaminase breaks down glutamine to glutamate and NH4+ in the liver
  • cycle repeats with glutamate and ammonia goes to urea cycle
  • alanine can be used to transport ammonia form the muscle to the liver
  • excess glutamine is processed in the intestines, kidneys, and liver
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53
Q

glucose-alanine cycle

A
  • vigorously working muscles operate nearly anaerobically and rely on glycolysis for energy
  • glycolysis yields pyruvate
  • if not eliminated, lactic acid will build up (sore)
  • pyruvate -> lactic acid
  • pyruvate take an amine from the glutamate in muscle and convert it to alpha-ketoglutarate and pyruvate becomes alanine (through alanine aminotransferase)
  • alanine is transported through blood and into liver
  • alanine is converted to pyruvate by alanine aminotransferase in the liver (alpha-ketoglutarate is converted to glutamate)
  • pyruvate gets converted to glucose through gluconeogenesis
  • glucose circulates and transported to muscle for energy
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54
Q

oxidative deamination leads to generation of ammonia

A
  • once glutamate gets the ammonia it must be broken down to release ammonia
  • glutamate dehydrogenase -mitochondrial enzyme (extracts H+)
  • NADP accepts H -> NADPH
  • glutamate is converted to alpha-iminoglutarate
  • in the presence of water it releases ammonia -> converted to alpha-ketoglutarate
  • can proceed in either direction, although it favors glutamate synthesis -> oxidative deamination
  • oxidative deamination takes place in mitochondrial matrix
  • can use either NAD+ or NADP+ as electron acceptor
  • ammonia is converted to urea for excretion
  • transdeamination- total pathway of accepting amino group from amino acid and releasing it inside the liver
  • pathway for ammonia excretion -> transdeamination = transamination + oxidative deamination
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55
Q

key points for ammonia generation

A
  • glutamine (from extrahepatic tissues), alanine (from muscle) mainly transport ammonia to the liver (deamination) for conversion to urea using the urea cycle
  • inside hepatocyte cytoplasm, alanine and other amino acids are converted to glutamate by transamination (using alpha-ketoglutarate as substrate)
  • glutamate is broken down by glutamate dehydrogenase
  • glutamine and glutamate can enter the mitochondria of the hepatocytes
  • inside the mitochondria:
  • glutaminase breaks down glutamine to ammonia and glutamate
  • glutamate dehydrogenase converts glutamate to ammonia and alpha-ketoglutarate by oxidative deamination
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56
Q

general urea cycle rxn

A
  • ammonia that is collected from glutamate and glutamine
  • occurs in liver (hepatocyte)
  • ammonia reacts with bicarbonate in the mitochondrial matrix
  • creates aspartate and 3 ATP
  • all these reactants react and from urea and fumarate
  • 5 enzymes involved (two mitochondrial and three cytosolic)
  • rxn requires 3 ATP
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57
Q

steps of the urea cycle: mitochondrial matrix

A
  • amino acids are converted to glutamate and glutamate is oxidatively deaminated to form ammonia by glutamate dehydrogenase (in the hepatic mitochondria)
  • glutamine is deaminated by glutaminase to form ammonia and glutamate -> oxydative deamination -> ammonia by glutamate dehydrogenase
  • carbamoyl phosphate synthetase 1 (CPS 1) takes the ammonia (and bicarbonate) and makes carbamoyl phosphate
  • carbamoyl phosphate is picked up by ornithine and converted to citrulline
  • citrulline goes outside of the mitochondrial matrix and into the hepatic cytosol
  • oxaloacetate (keto acid) can also take the amino group from glutamate (transamination) and form aspartate which will be used later on
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58
Q

carbamoyl phosphate synthetase 1 (CPS 1)

A
  • in the hepatic mitochondrial matrix
  • rate limiting step
  • picks up ammonia and bicarbonate and converts it into carbamoyl phosphate
  • uses 2 ATP
  • uses the first ATP molecule that interacts with the bicarbonate -> ADP + carbonic-phosphoric acid anhydride
  • ammonia binds to carbonic-phosphoric acid anhydride and forms carbamate
  • 2nd ATP is used and forms carbamoyl phosphate
  • this is the 1st nitrogen-acquiring rxn of the urea cycle
  • ammonia travels through a tunnel in CPS 1 (encapsulated)
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59
Q

nitrogen from carbamoyl phosphate enter the urea cycle

A
  • majority of rxns within urea cycle occur in cytosol
  • ornithine is carbomoylated to make citrulline by ornithine transcarbamoylase in mitochondria
  • citrulline can enter cytosol
  • argininosuccinate synthetase takes 2nd nitrogen from aspartate (aspartate was made in mitochondrial matrix) and uses an ATP -> produces argininosuccinate
  • argininosuccinase breaks argininosuccinate into fumarate and arginine
  • argininosuccinase eliminates fumarate to form arginine
  • arginase liberates urea from arginine
  • ornithine is regenerated in the last process and recycled
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60
Q

urea cycle

A

aka ornithine cycle

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61
Q

citrulline + aspartate

A
  • using ATP form the backbone of argininosuccinate

- process is done by argininosuccinate synthetase

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62
Q

urea

A

-contains the amino group from glutamate and aspartate

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63
Q

regulation of the urea cycle: NAG

A
  • elevated levels of arginine -> activates N-acetylglutamate synthase
  • N-acetylglutamate synthase makes the reaction between acetyl-CoA and glutamate
  • N-acetylglutamate synthase converts to N-acetylglutamate (NAG)
  • NAG binds to CPS 1 and causes conformational changes that opens up the tunnel that links 2 of the active sites
  • CSP 1 is activated by N-acetylglutamate (NAG)
  • expression of urea cycle enzymes increases when needed -> high protein diet (we cant store amino acids), starvation (when protein is being broken down for energy), diabetes mellitus
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64
Q

Aspartate-arginosuccinate

A
  • shunt (krebs bicycle) links urea cycle and citric acid cycle
  • malate-aspartate shuttle connects the urea and citric acid cycle
  • aspartate-argininosuccinate shunt in which fumarate produced in the urea cycle can be used to make aspartate in the mitochondrial matrix, thereby linking the urea cycle to the citric acid cycle
  • argininosuccinate is converted to fumarate -> fumarate is converted to malate -> malate is used in citric acid cycle
  • fumarate is the precursor of malate
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65
Q

key points for urea cycle

A
  • occur in the liver (hepatocyte)
  • nitrogen atoms come from glutamate and aspartate
  • even glutamine is converted to glutamate before oxidative deamination for ammonia generation
  • CPS 1 uses 2 ATP and makes use of substrate channeling to protect labile intermediates
  • after CPS 1, the cycle has 4 enzymatic steps (1 in mitochondria and 3 in cytosol) -> uses 1 more ATP
  • 2 mitochondrial and 3 cytosolic enzymes participate in urea cycle
  • rates of these enzymes are controlled by concentration of substrate
  • overall rate can be controlled by glutamate levels
  • products are fumarate and urea
  • urea is excreted in urine by the kidneys
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66
Q

memorize the amino acids

A
  • aliphatic- alanine, glycine, isoleucine, leucine, proline, valine
  • aromatic- phenylalanine, tryptophan, tyrosine
  • acidic- aspartic acid, glutamic acid
  • basic- arginine, histidine, lysine
  • hydroxylic- serine, threonine
  • sulfur-containing- cysteine, methionine
  • amidic- asparganine, glutamine
  • essential- Ile, leu, val, his, trp, phe, lys, thr, met
  • non-essential- ala, gly, pro, tyr, asp, glu, arg, ser, cys, asn, gln
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67
Q

essential vs. nonessential and conditionally essential amino acids

A
  • essential amino acids must be obtained as dietary protein -> cannot be synthesized by humans
  • nonessential amino acids are easily synthesized from central metabolites
  • conditionally essential- can only be made at certain times of life, required to some degree in young, growing animals and/or sometimes during illness
  • consumption of a variety of foods supplies all the essential amino acids
  • essential- Ile, leu, val, his, trp, phe, lys, thr, met
  • non-essential- ala, asp, glu, ser, asn
  • conditionally essential- arg, cys, gln, gly, pro, tyr
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68
Q

overview of amino acid catabolism

A
  • amino acids broken down
  • go through transamination and oxidative deamination
  • ammonia -> urea
  • carbon skeleton is passed onto krebs cycle/citric acid cycle
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69
Q

fate of carbon skeleton

A

-synthesis of lipids, glucose or production of energy through their oxidation to CO2 and H2O

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70
Q

catabolism of amino acids

A
  • different amino acids carbon skeleton gets directed to central metabolic pathway
  • oxidative breakdown of amino acids can acount for 10-15% of our metabolic energy
  • occurs primarily in muscle or liver
  • intermediates of the central metabolic pathway
  • some amino acids result in more than one intermediate
  • glucogenic amino (18) acids can be converted to glucose
  • ketogenic amino acids (7) can be converted to ketone bodies
  • some are both (5)
  • leucine and lysine are only ketogenic
  • is an amino acid is converted to acetyl-coA or acetoacetyl-CoA -> it will become a ketone body
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71
Q

arg, glut, his, pro

A
  • form glutamate
  • convert to alpha-ketoglutarate
  • glucose
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72
Q

ile, met, thr, val

A
  • forms succinyl-CoA

- glucose

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73
Q

phe, tyr

A
  • form fumarate

- glucose

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74
Q

asp,asn

A

form oxaloacetate

-glucose

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75
Q

ala, cys, gly, ser, thr, trp

A
  • form pyruvate

- can form ketone or glucose

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76
Q

ile, leu, thr, trp, lys, phe, tyr

A
  • form ketone bodes

- acetyl- CoA and acetoacetyl CoA

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77
Q

glucogenic amino acids

A
  • based on the intermediates produced during their catabolism
  • amino acids that can be converted into glucose through gluconeogenesis
  • amino acids whose catabolism yields pyruvate or one of the intermediates of the citric acid cycle are termed glucogenic or glycogenic
78
Q

ketogenic

A
  • based on the intermediates produced during their catabolism
  • amino acids that can be converted into ketone bodies through ketogenesis
  • amino acids whose catabolism yields either acetoacetate or one of its precursor (Acetyl-CoA or acetoacetyl-CoA) are termed ketogenic
79
Q

ketone bodies

A
  • 3 water soluble ketones
  • by-products of fatty acid oxidation
  • when fatty acids are broken down for energy in the liver and kidney -> generate ketone bodies
  • 3 ketone bodies are acetone, acetoacetic acid, and beta hydroxybutyric acid
  • beta-hydroxybutyric acid- doesnt have the ketone carbonyl group
  • ketone bodies are transported from the liver to other tissue where acetoacetate and beta-hydroxybutyrate can be reconverted to acetyl-CoA to produce energy via krebs cycle
  • excess ketone bodies accumulation is called ketosis
80
Q

glucogenic only

A 
NONESSENTIAL
-ala
-arg
-asn
-cys
-glu
-gln
-gly
-pro
-ser
ESSENTIAL
-his
-met
-thr
-val
81
Q

glucogenic and ketogenic

A
  • tyr- nonessential

- ile, phe, trp- essential

82
Q

ketogenic only

A
  • leu and lysine

- essential

83
Q

cofactors involved in amino acid catabolism

A
  • important in 1-carbon transfer rxns -> tetrahydrofolate (THF) (vitamin b9)
  • most are derived from vitamin b complex
  • pyridoxal-5-phosphate (PLP) -> vitamin B6
  • biotin -> vitamin B7 -> one carbon transfer
  • S-adenosylmethionine -> one carbon transfer
  • 1 carbon transfer- adds a single carbon
84
Q

pyridoxal phosphate (PLP)

A
  • co-factor
  • derived from pyridoxine
  • aldehyde form- pyridoxal phosphate
  • aminated form- pyridoxamine phosphate (can react with carbonyl)
  • enzyme- enzyme-PLP schiff base -> amino acid metabolism
85
Q

biotin

A
  • co-factor
  • single carbon comes from CO2 in the form of bicarbonate
  • use ATP hydrolysis to attach to C
  • carboxylates
  • becomes carboxylated
  • biotin carboxylase (BC) can easily give up its extra single carbon
  • when it gives up its carbon to acetyl-CoA it converts back to biotin and acetyl-CoA becomes malonyl-CoA
  • transfer to acetyl-CoA uses carboxyltransferase (CT)
86
Q

tetrahydrofolate (THF) as a co-factor/co-enzyme

A
  • THF is important in one carbon transfer in different oxidation states
  • addition of oxygen or removal of H -> oxidation
  • addition of H and removal of O -> reduction
  • transfers CH3 (reduced), CH2OH, and CHO (oxidized)
  • N5 or N10 or both are used for carbon transfer
  • used in a wide variety of metabolic rxns
  • provides one carbon donors of different oxidation states
  • carbon generally comes from serine
  • there are freely interconvertible in the folate cycle
  • the C1 units are attached to THF at the N5 or N10 or both positions
  • NAD+ accepts H and oxidizes the rxn
  • NADH donates a H and reduces the rxn
  • formate- simple donor- can attach to N10 -> N10-formyl-THF or N5 -> N5-Formyl-THF
  • bidirectional
87
Q

NAD+ accepts and H

A
  • oxidizes the reaction for carbon transfer

- if it donates a H it reduces the rxn

88
Q

N5-methyl-THF

A

-amino acids biosynthesis of Methionine

89
Q

N5,N10-methylene-THF

A
  • amino acid degradation

- SHMT- Ser to Gly

90
Q

N10-formyl-THF

A

-purine synthesis

91
Q

-S-adenosylmethionine (adoMet) as co-factor/co-enzyme

A
  • adoMet is the preferred cofactor for methyl (CH3) transfer in biological rxn
  • methionine and ATP react and form SAM or adoMet -> this can give up methyl group -> forms S-adenosylhomocysteine -> broken down into AMP -> forms homocysteine
  • homocysteine can pick up N5-methyl-THF and form methionine again (repeat)
  • SAM (adoMET) is 1000 times more reactive than THF methyl group
  • synthesized from ATP and methionine
  • regeneration uses N5-methyl-THF (only known use in mammals)
92
Q

end products of amino degradation

A
  • 7 of the amino acids are ketogenic and convert to acetyl-CoA or acetoacetyl-CoA (leu, ile, thr, lys, phe, tyr, trp)
  • 6 amino acids get converted to pyruvate (ala, cys, gly, ser, thr, trp)
  • 5 amino acids to alpha-ketoglutarate (arg, glu, gln, his, pro)
  • 4 amino acids to succinyl-CoA via propionyl-CoA (ile, met, thr, val)
  • 2 amino acids convert to fumarate (phe, tyr)
  • 2 amino acids convert to oxaloacetate (asp, asn)
  • all of these are ketoacids other than succinyl-CoA and fumarate (and acetyl-CoA)
93
Q

degradation of amino acids to pyruvate

A

-6
-ala, cys, gly, ser, thr, trp
-alpha-ketoacid accept amino group from alanine via alanine aminotransferase (PLP) -> glutamate
-alanine loses amino group -
> pyruvate
-pyruvate can be converted to glucose through gluconeogenesis
-Thr is converted to glycine
-serine hydroxymethyltransferase (SHMT (N5,N10 transfer)) converts Gly to Ser using PLP (adds a carbon)
-gly and thr get converted to ser
-Trp is first converted to Ala in few steps
-ser, ala, cys get directly converted to pyruvate
-serine dehydratase converts ser to pyruvate (PLP)
-alanine is converted to pyruvate by aminotransferase using PLP-dependent rxns
-cys is converted to pyruvate within a few steps

94
Q

degradation of Asn and Asp to oxaloacetate

A
  • 2 -> asp, asn
  • asparagine is first converted to aspartate by asparaginase
  • alpha-ketoglutarate accepts amino group from aspartate (PLP-dependent transamination rxn by aspartate aminotransferase)
  • oxaloacetate is formed and converted to glucogenic through gluconeogenesis
95
Q

degradation of amino acids to alpha-ketoglutarate

A
  • 5 -> arg, glu, gln, his, pro -> converted to glu first
  • part of urea cycle
  • common byproducts- urea, ammonia, glu
  • ammonia (NH3) generated is toxic and can be converted to ammonium ion and excreted or combined with glu to make gln by glutamine synthetase
  • arginine is broken down by arginase and is converted to urea -> converts to ornithine (key part of urea cycle)
  • all of the amino acids go through steps and convert to glutamate
  • glutamate is deaminated (oxidative) to alpha-ketoglutarate via glutamate dehydrogenase (GDH) (NAD -> NADH oxidizes and and accepts amino group)
  • alpha-ketoglutarate goes through krebs cycle -> gluconeogenesis -> glucose
96
Q

degradation of branched-chain amino acids

A
  • leucine, isoleucine and valine are oxidized for fuel
  • occurs in muscles, adipose tissue, the kidneys, and the brain
  • aminotransferase DOES NOT occur in liver
  • amino groups are removed through transamination and accepted by alpha-ketoglutarate (by branched chain aminotransferase PLP-dependent)
  • the amino acids are now ketoacids and are converted to acyl-CoA derivatives through branch-chain alpha-keto acid dehydrogenase complex (BVKDH)
  • during this reaction the ketocids lose a carbon in the form of CO2 -> coenzyme A attaches where the carbon was and becomes oxidized by NAD
  • final products: acetyl-CoA (ile, leu), succinyl-CoA (ile, val) and acetoacetate (leu)
97
Q

maple syrup urine disease

A
  • BCKDH- mutated in this disease
  • branched chain alpha-keto acid dehydrogenase is mutated
  • BCKDH converts ketoacids into acyl-CoA derivatives during branched chain amino acid degradation
  • build up of keto acids
  • rare autosomal recessive disease
  • manifests in the 1st few weeks of life
  • leads to neurological deterioration, mental and physical retardation
  • can cause seizures, muscular tension and coma
  • urine smells like maple syrup or burnt sugar
98
Q

degradation of branched chain amino acids Met, Lle, Thr, and Val to succinyl-CoA

A
  • 4 -> ile, met, thr, val
  • succinyl-CoA is not a keto acid
  • degradation takes place in extrahepatic tissues
  • succinyl-CoA always derived from propionyl-CoA
  • amino acids go through reactions and finally propionyl CoA is converted to succinyl-CoA
  • succinyl-CoA goes through krebs cycle -> gluconeogenesis -> glucose
  • methionine needs adoMet to convert
99
Q

degradation of ketogenic amino acids

A
  • 2 -> leu and lys
  • only ketogenic
  • final product is acetyl-CoA from acetoacetyl-CoA
  • lysine and leucine -> acetoacetyl-CoA -> acetyl-CoA
  • trp, tyr, phe, ile can also take this pathway but they can also take a different pathway that is glucogenic
100
Q

genetic defects in many steps of Phe degradation lead to disease

A
  • in phenylketonuria (PKU) (a disease):
  • if there is a mutation in phenylalanine hydroxylase it wont be able to convert to tyrosine -> it will then take a diff pathway that makes phenylpyruvate and phe
  • a buildup of phe and phenylpyruvate
  • phenylpyruvate accumulates in the tissues, blood, and urine
  • impairs neurological development leading intellectual deficits
  • controlled by limiting dietary intake of phe
101
Q

amino acids are the building blocks for numerous biological important molecules: tryptophan

A
  • tryptophan is converted into nicotinate (niacin)
  • nicotinate (niacin)- precursor for NAD
  • NAD/NADP- helps in absorption of H or release of H (oxidation or reduction)
  • tryptophan can also form serotonin
  • tryptophan can also form indoleacetate, a plant growth factor
102
Q

amino acids are the building blocks for numerous biological important molecules: glutamate

A
  • gamma amino butyric acid (GABA) is a mjor neurotransmitter in the CNS
  • derived from glutamate
  • important for neurodevelopment
103
Q

amino acids are the building blocks for numerous biological important molecules: histamine

A
  • Histamine released by mast cells or basophils and initiate the inflammatory or allergic response
  • derived from histidine
104
Q

amino acids are the building blocks for numerous biological important molecules: tyrosine

A

-melanin, dopamine, and epinephrine are derived from tyrosine

105
Q

summary of amino acid catabolism/fate of carbon skeleton

A
  • thr is also ketogenic as it can get converted to acetyl-coA (by alternate pathway)
  • leucine and lysine are only ketogenic
  • amino acids from protein are an important energy source in carnivorous animals
  • 1st step of amino acid catabolism is transfer of the NH3 via PLP-dependent aminotransferase usually to alpha-ketoglutarate to yield L-glutamate
  • most mammals, toxic ammonia is quickly recaptured into carbamoyl phosphate and passed into the urea cycle
  • amino acids are degraded to pyruvate, acetyl-CoA, alpha-ketoglutarate, succinyl-CoA, and/or oxaloacetate
  • amino acids yielding acetyl-CoA are ketogenic
  • amino acids yielding other end products are glucogenic
  • genetic defects in amino degradation pathways result in a # of human diseases
  • amino acid catabolism is dependent on a variety of cofactors, including THF, ado-Met, and PLP
106
Q

biochemistry of molecular nitrogen

A
  • atmosphere is 80% N2 but it is in non useful form
  • N2 is mostly chemically inert and plants and animals cant directly make use of atmospheric nitrogen
  • nitrogen is an important constituent of amino acids (proteins), nucleotides and nucleic acids and other essential biomolecules
  • the biologically useful form of nitrogen is ammonia (NH3)
  • N2+ 3 H2 -> 2 NH3
  • energetically favorable
  • even though ΔG’ = -33.5kJ/mol… breaking a triple bond has high activation energy (hard to break bond) -> forms amino acids
  • can be converted using nonbiological process:
  • N2 and O2 -> NO (via lightening)
  • N2 and H2 -> (via the industrial Haber process -> requires tempature >400C, pressure > 200atm)
107
Q

nitrogen cycle

A
  • chemical transformations maintain a balance between N2 (gaseous) and biologically useful forms of nitrogen
  • plants can take up nitrogen and make proteins -> eaten
  • atmospherical nitrogen (gas) gets trapped by microbes in plants -> convert to NH3
  • nitrogen fixing bacteria -> nitrates (NO3-) or nitrites (NO2-) -> taken up by plants -> NH3 -> animals eat
  • decomposition releases ammonia back into soul
  • denitrifying bacteria- degrades and breakdowns ammonia (oxidizes) back into gas form
  • total amount of nitrogen fixed annually in the biosphere exceeds 10^11kg
108
Q

fixation

A
  • done by bacteria (Diazotrophs) that reduce N2 to NH3/NH4+
  • reduce nitrogen (gas) to ammonia or ammonium
  • anaerobic
  • direct
109
Q

nitrification

A
  • bacteria oxidize ammonia into nitrite (NO2-) and nitrate (NO3-)
  • Add oxygen and remove H
110
Q

assimilation

A
  • plants and microorganisms reduce NO2- and NO3- to NH3 via nitrite reductases and nitrate reductases
  • aerobic
  • NH3 is incorporated into amino acids, and so on
  • animals eat the plants
  • organisms die and plant/animal waste returns nitrogen to the soil as NH3
  • nitrifying bacteria again convert NH3 to nitrite and nitrate
111
Q

denitrification

A
  • nitrate is reduced to N2 under anaerobic conditions
  • NO3- (nitrate) is the ultimate electron acceptor instead of O2
  • reduction; therefore, we dont want oxygen present
112
Q

assimilation: nitrate reductase: step 1

A
  • reduces
  • NO3- (nitrate) +2e- -> NO2- (nitrite)
  • large, soluble protein
  • contains novel Mo cofactor
  • e- from NADH
113
Q

assimilation: nitrite reductase: step 2

A
  • NO2- + 6e- -> NH4+ (ammonia)
  • complete loss of O -> reduced
  • carried out and found in chloroplasts in plants: e- comes from ferredoxin
  • takes unusable nitrate and nitrites in soil and makes ammonia
  • in non photosynthetic microbes: e- comes from NADPH
114
Q

some organisms can directly fix N2 to useful forms

A
  • most are single celled prokaryotes (archaea)
  • some live in symbiosis with plants (proteobacteria with legumes such as peanuts, beans)
  • a few live in symbiosis with animals (spirochete with termites)
  • gives animal ammonium and the spirochete takes nutrients from host
  • they have enzymes that overcome the high activation energy (of breaking N bonds) by binding and hydrolyzing ATP
115
Q

Haber process

A
  • industrial process used to make ammonia
  • power plants
  • N2 + 3 H2 -> 2 NH3 (ΔH = -92.4 kJ/mol)
  • fertilizer made from this process sustains 1/3rd of the earth population
  • used to farm crops
  • process consumes 1-2% of the worlds annual energy supply
116
Q

diazatrohps

A
  • naturally makes ammonia
  • convert N2 to NH3 using nitrogenase*
  • fixes nitrogen
  • this is where we get all our amines for amino acid synthesis
  • a very high energy (ATP consuming) process
  • downstream, glutamate synthase incorporates the NH3 into glutamate
117
Q

nitrogen fixation is carried out by the nitrogenase complex

A
  • N2 + 3 H2 -> 2 NH3
  • exergonic (ΔG = -33.5 kJ/mol) reaction but very slow due to the triple bonds high activation energy
  • Eact= 230 -420 kJ/mol
  • even though it is spontaneous -> super slow and hard to do bc of the high activation energy
  • the nitrogenase complex uses ATP to overcome the activation energy
  • passes electrons to N2 and catalyzes a step-wise reduction of N2 to NH3
  • N2 + 8 H+ + 8e- +16 ATP -> 2 NH3 + H2 + 16ADP + 16Pi
  • 2NH3 + 2 H+ -> 2 NH4+ (ammonia gets protonated to ammonium ion)
  • about 16 ATP molecules are consumer per one molecule of N2 -> makes 2 ammonia
  • ATP consuming
118
Q

nitrogenase complex

A
  • a large protein complex involved in reducing nitrogen
  • ATP hydrolysis and ATP binding help overcome the high activation energy
  • has a dinitrogenase reductase domain, alpha and beta dinitrogenase -> tetramer
  • source of e- varies between organisms (often pyruvate -> ferredoxin)
  • makes use of several metal cofactors:
  • 4Fe-4S cluster
  • P cluster
  • FeMo cluster
  • makes use of these metal cofactor cluster to transport these e- through the molecule for reduction of nitrogen
  • metal cofactors act as “wires” to transfer -> not directly connected but when an e- comes and accepts it releases another one -> goes onto the next cluster
  • enzyme requires anaerobic environment -> oxidation destroys the metal cofactors
119
Q

nitrogen fixation by the nitrogenase complex

A
  1. pyruvate passes 8e- to ferredoxin or flavodoxin -> gets reduced
  2. ferredoxin or flavodoxin pass 8e- to dinitrogenase reductase -> gets reduced
    - uses 16 molecules of ATP hydrolysis
  3. the reductase passes 8e- to dinitrogenase
  4. dinitrogenase passes 6e- to nitrogen (or to protons) and reduces it to make 2 NH3
    - the other 2e- reduce the hydrogen and make H2
  5. formation of H2 appears an obligatory side reaction
    - byproduct
120
Q

redox reactions in nitrogenase

A
  • the net reaction of the nitrogenase complex
  • N2 +8 H+ + 8e- + 16 ATP -> 2 NH3 + H2 + 16 ADP + 16 Pi
  • dinitrogenase reductase catalyzes: transfer of 8e- to dinitrogenase and hydrplysis of ATP with release of protons
  • dinitrogenase catalyzes: transfer of 6e- to nitrogen -> formation of 2 NH3 and transfer of 2e- to protons -> formation of H2
  • mechanism of dinitrogenase is poorly understood
  • in reality about 20-30 ATP molecules are used per N2 in the cell -> not effective
121
Q

diazotrophs x leguminous plant

A
  • fix nitrogen
  • plant takes care of ATP and O2 lability
  • bacteria has access to plants carbohydrate and citric acid cycle intermediates for energy
  • bacteria are covered with leghemoglobin to bind O2 and prevent corruption of the catalyst nitrogenase (nitrogenase is anaerobic) -> maintains anaerobic environment
  • bacteria live in the root nodules of the plant
  • fix nitrogen by nitrogenase
  • bacteria and plant have a symbiotic relationship
  • bacteria gets the ATP from the plant to fix the nitrogen (high ATP demand)
  • plant gets the ammonium from the bacteria
  • produces more NH3 than the plant needs
  • excess NH3 is released into the soil
  • rhizobium- the bacteria
122
Q

nitrogen assimilation vs. nitrogen fixation

A 
Nitrogen Assimilation:
-converts NO3- or NO2- to NH3
-uses electrons from NADH, NADPH, or photosynthetic transfer from ferrodoxin
-does not require as much ATP
Nitrogen Fixation:
-converts N2 to NH2
-requires multiple ATP uses electrons from pyruvate
-huge energy requirement
BOTH:
-are electron transfer processes
-use Mo cofactor
-involve multiple redox cofactors, such as Fe-S, NADH, NADPH, ferrodoxin, flavodoxin...etc.
123
Q

ammonia is incorporated into biomolecules through Glu and Gln

A
  • glutamine is made from glutamate by glutamine synthase in a 2-step process
  • uses ATP hydrolysis -> converted to gamma-glutamyl phosphate -> phosphate is displaced by ammonia that was fixed -> forms glutamine
  • phosphorylation of glutamate creates a good leaving group that can be easily displaced by ammonia
  • glutamate has one ammonia group while glutamine has two -> second group comes from the ammonia that is fixed which is added to gamma-glutamyl phosphate
  • this process is carried out by the ring structure of glutamine synthetase (12 units)- converts glutamate into glutamine
124
Q

glutamine synthetase and glutamate synthase

A

GLUTAMINE SYNTHETASE:
-ammonia taken up by glutamine synthetase to make glutamine
-glutamate + NH4+ + ATP -> glutamine + ADP +Pi +H+
GLUTAMATE SYNTHASE:
-glutamate synthase makes glutamate (bacteria and plants; animals do not have glutamate synthase, but use transamination and other pathways for glutamate)
-glutamine + alpha-ketoglutarate + NADPH + H+ -> glutamate + glutamate + NADP+
COMBINED RXN:
-alpha-ketoglutarate + NH4+ + NADPH + ATP -> glutamate + NADP+ + ADP + Pi
-overall reaction that can happen in bacteria
-glutamate can also be made using alpha-ketoglutarate via glutamate dehydrogenase (reverse reaction to urea cycle)
-in humans: reverse rxn in the urea cycle can form glutamate from alpha-ketoglutarate by glutamate dehydrogenase

125
Q

amino acid biosynthesis

A
  • takes place in every living organism (we focus on humans)

- consumption of a variety of food supplies all the essential amino acids

126
Q

biosynthesis of amino acids: key points

A
  • many of the same rxns from catabolism of amino acid
  • glutamine is a key regulator and control point (absorption of ammonia) -> helps transfer amine group
  • substrate for other pathways
  • levels can be regulated by glutamine synthetase
  • glutamine synthetase can be regulated (allosterically) by the products of pathways that start with or require glutamine (negative feedback)
  • not all pathways operate in all organisms
  • Ile, Leu, and Val (essential AA’s) and some others can be synthesized in plants and many prokaryotes
127
Q

amino acid synthesis overview

A
  • source of amino group for amino acid biosynthesis is glutamate or glutamine
  • amino acids (backbone) can be derived from intermediates of:
  • glycolysis (3-phosphoglycerate, phosphoenolpyruvate, pyruvate)
  • citric acid cycle (oxaloacetate, alpha-ketoglutarate)
  • pentose phosphate pathway (erythrose 4-phosphate, ribose 5-phosphate)
  • bacteria can synthesize all 20 amino acids
  • mammals cant synthesize all amino acids and require some from diet (essential amino acid)
128
Q

precursor: alpha-ketoglutarate

A
  • glutamate
  • glutamine
  • proline
  • arginine
129
Q

precursor: pyruvate

A
  • alanine
  • valine
  • leucine
  • isoleucine
130
Q

precursor: 3-phosphoglycerate

A
  • serine
  • glycine (derived from serine)
  • cysteine
131
Q

precursor: phosphoenolpyruvate and erythrose 4-phosphate

A
  • tryptophan
  • phenylalanine
  • tyrosine
132
Q

precursor: oxaloacetate

A
  • aspartate
  • asparagine
  • methionine
  • threonine
  • lysine
133
Q

precursor: ribose 5-phosphate

A

-histidine

134
Q

proline and arginine dervive from glutamate

A
  • come from alpha-ketoglutarate (krebs/citric acid cycle)
  • alpha-ketoglutarate -> glutamate -> glutamine, proline, arginine
  • amino acid gives up amino group to alpha-ketoglutarate
  • alpha-ketoglutarate becomes glutamate
  • amino acid becomes alpha keto acid
  • transamination -> PLP-dependent
  • glutamine synthetase makes glutamine form glutamate (adds an extra ammonium into glutamate -> glutamine)
  • different mechanisms from glutamate to pro and ala
135
Q

biosynthesis of proline from glutamate

A
  • inside bacteria glutamate get converted to gamma-glutamyl phosphate -> glutamate gamma- semialdehyde -> eventually becomes P5C (ring form) -> converted to proline
  • in humans ornithine is derived from urea cycle or degradation of arginine
  • ornithine gamma-aminotransferase converts ornithine to glutamate gamma-semialdehyde -> P5C -> cyclized and converts to proline (same last steps as bacteria)
  • same as bacteria but the precursor for humans is ornithine and for bacteria it is glutamate
136
Q

arginine is synthesized from ornithine in animals

A
  • ornithine comes from the urea cycle from the degradation of arginine
  • the rxn process in the inverse direction for synthesis of arginine from ornithine
  • to get back to arginine we go in reverse
  • ornithine -> associates with carbamoyl phosphate -> converted to citrulline -> converted to argininosuccinate through association with ATP and aspartate -> reacts with fumarate -> converts to arginine
137
Q

serine derives from 3-phosphoglycerate of glycolysis

A
  • same pathway in all organisms
  • requires glu as source of NH3 group
  • oxidation -> transamination -> dephosphorylation to yield serine
  • 3-phosphoglycerate -> glutamate -> transamination from alpha-ketoglutarate -> dephosphorylated-> serine -> tetrahydrofolate rxn -> glycine
  • tetrahydrofolate rxn- carbon is removed
138
Q

glycine derived from serine (serine derived from 3-phosphateglycerate of glycolysis)

A
  • carbon removed using tetrahydrofolate (THF)
  • reaction uses serine hydroxymethyltransferase (SHMT)
  • serine loses the hydroxymethyl group -> forms glycine
  • carbon is removed from serine using THF -> converts to glycine
139
Q

biosynthesis of cysteine from homocysteine and serine in mammals

A

-homocysteine reacts with serine -> forms sulfide linage cystathionine -
> PLP breakdown of cystathionine -> cysteine and alpha-ketobutyrate
-serine accepts the sulfhydryl group from homocysteine
-in mammals, sulfur is recycled from methionine degradation

140
Q

oxaloacetate yields aspartate, which yields asparagine, methionine, lysine, and threonine

A
  • aspartate is formed from transamination of oxaloacetate
  • aspartate is the root of the other amino acids
  • oxaloacetate -> aspartate -> asparagine, methionine, lysine, threonine
  • recall: pyruvate -> alanine, valine, leucine, isoleucine
  • aspartate gets converted from oxaloacetate through transamination
141
Q

pyruvates yields alanine, valine, leucine, and isoleucine

A
  • pyruvate -> alanine, valine, leucine, isoleucine
  • pyruvate to alanine involves amino transferase rxn transamination
  • alanine formed through breakdown process of pyruvate
  • simple starting material but non-trivial synthesis
  • multiple intermediates and multiple enzymatic rxns involved using several cofactors
  • pyruvate interacts with TPP (thyominepyrophosphate) and forms resonance stabilized molecules -> interacts with another pyruvate -> valine, leucine
  • at the resonance stabilized molecule step the rxn can go in a different pathway that forms isoleucine
  • forms branched amino acids
142
Q

amino acid biosynthesis can be inverse of catabolic pathways

A
  • pyruvate -> aminotransferase transamination -> alanine
  • oxaloacetate -> add amino from transamination -> aspartate -> add amino from glutamine -> asparagine
  • alpha-ketoglutarate -> ass amino from transamination -> glutamate -> add amino from glutamine synthetase -> glutamine
  • all reversible
143
Q

aromatic acids derive from phosphoenolpyruvate and erythrose 4-phosphate

A
  • very complicated chemistry
  • rings must be synthesized and closed and then oxidized to create double bonds
  • phosphoenolpyruvate and erythrose-4-phosphate: aromatic
  • hardly happens in humans
  • chorismate is a common intermediate
  • chorismate is converted to phenylalanine, tyrosine, tryptophan
  • phenylalanine -> tyrosine
144
Q

glutamine is a key central player in amino acid biosynthesis

A
  • glutamine is an amino donor for several rxn in amino acid biosynthesis
  • glutamine also is an important substrate for other metabolic rxns (nucleotide metabolism, for example)
  • glutaime -> converted to oxaloacetate -> aspartic acid -> asparagine
  • glutamine levels can be regulated by glutamine synthetase
  • glutamine synthetase, thus, plays an important role in regulating nitrogen levels in the cell (and amino acids)
  • highly regulated
  • maintains amino acid pool
145
Q

glutamine synthetase regulation

A

-much more known about bacterial system of glutamine regulation
-can be regulated allosterically by:
-histidine
-tryptophan
-carbamoyl phosphate
-glucosamine-6-phosphate
-AMP
-CTP
-these are products that glutamine makes that can actually block or enhance the production of glutamine in excess
-all end products of pathways starts with glutamine
-also regulated by ala, ser, and gly (sensors of the cells nitrogen levels)
-

146
Q

nitrogen cycle and amino acid biosynthesis summary

A
  • methods for activation of molecular nitrogen to nitrates, nitrites, and ammonia
  • glutamine serves as the primary ammonia donor in amino acid biosynthesis
  • 20 common amino acids are synthesized from alpha-ketoglutarate, 3-phosphoglycerate, oxaloacetate, pyruvate, phosphoenolpyruvate, erythrose 4-phosphate, and ribose-5-phosphate
  • essential amino acids need to be obtained from dietary sources and can be converted to other amino acids or biomolecules
  • we cannot make all the amino acids (non-essential)
147
Q

nucleoside

A
  • made up of nitrogenous bases
  • pentose sugar
  • pyrimidine
  • purine- double ring
  • purine or pyrimidine base associates with pentose sugar and phosphate attaches -> nucleotide
148
Q

nucleotide

A
  • nitrogenous base
  • pentose sugar
  • phosphate
  • nucleoside with a phosphate attached to the 5’ carbon of pentose (can be mono, di, tri)
  • carbon and nitrogen atoms on the nitrogenous base are numbered in cyclic format
  • carbons of the pentose sugar are designated N’ (prime) to prevent confusion
  • the 5’-phosphate esters of nucleosides
149
Q

purine and pyrimidines

A
  • nitrogenous bases (nucleobases) that are a component of nucleosides, nucleotides, and nucleic acids
  • pyrimidine- one ring -> cytosine, thymine, uracil
  • purine- two ring -> adenine, guanine
150
Q

nucleosides

A
  • N-linked glycosides
  • bases of the nucleosides are connected to the sugar through N-linked glycosides
  • bonded to anomeric carbon of pentose sugar
151
Q

nucleic acids

A

-polymers of nucleotides

152
Q

functions of nucleotides and nucleic acids

A
  • nucleic acids:
  • storage of genetic info (DNA)
  • transmission of genetic info (mRNA)
  • processing of genetic information (ribozymes)
  • protein synthesis (tRNA and rRNA)
  • nucleotides are also used in the monomer form for cellular functions:
  • energy for metabolism (GTP, ATP)
  • enzyme cofactors (CoA, FAD, NAD+)
  • signal transduction (cGMP, cAMP) -> cyclic
  • neurotransmission (adenosine)
153
Q

deoxyribonucleotides

A
  • 2’ carbon in this ribose sugar doesnt have OH group
  • loses one of its OH groups and replaces with H
  • made up of polymers of deoxyribosnucleotides
  • present in DNA
154
Q

ribonucleotides

A
  • ribose has OH groups on the 2’ carbon

- present in RNA

155
Q

nucleotide biosynthesis

A
  • nucleotides can be synthesized de novo (from the beginning) from amino acids, ribose-5-phosphate, CO2, and NH3
  • amino acids that lead to purine nucleotides: glycine, glutamine, aspartic acid
  • nucleotides can be salvaged from RNA, DNA, and cofactor degradation (sugars and nucleotides are recycled and reused once broken down) -> quicker
  • many parasites (plasmodium sp., trypanisoma brucei) lack de novo biosynthesis pathways and rely exclusively on salvage
  • compounds that inhibit salvage pathways are promising antiparasitic drugs
156
Q

salvage vs. de novo biosynthesis

A
  • both purines and pyrimidines can be made via the salvage (recycling) pathway
  • put together a preformed nucleobase and sugar to make the nucleotides
  • one step pathway
  • low energy (no ATP required)
  • this is the predominant pathways under ‘normal’ conditions
  • why do we need another way to make nucleotides? (de novo) -> during high nucleotide demand, like cell-division
  • drugs that block de novo pathway are anticancerous bc cancer has rapid cell division
157
Q

de novo biosynthesis of nucleotides

A
  • the same in all organisms (mostly)
  • bases synthesized while attached to ribose (NOT independently made and combined at end)
  • built on the sugar molecule
  • Gln provides most amino groups
  • Gly is precursor for purines
  • Asp is precursor for purines and pyrimidines
  • nucleotide pools are kept low, so cells must continually synthesize them during high demand events
  • this synthesis may limit rates of transcription and replication
158
Q

5-Phosphoribosyl-1-Pyrophosphate (PRPP) is the precursor for the pentose sugar

A
  • synthesized from ribose 5-phosphate of pentose phosphate pathway via ribose phosphate pyrophosphokinase
  • ribose sugar with phosphate attached to 5’
  • pyrophosphate is attached to 1’
  • foundation for nitrogenous base for de novo synthesis
159
Q

overview of biosynthesis of nucleotides (purine/pyrimidine)

A
  • PRPP -> 11 steps (de novo) -> IMP -> purine nucleotide synthesis
  • PRPP + base -> salvage step (only 1) -> IMP -> purine nucleotide synthesis
  • PRPP -> 6 steps (de novo) -> UMP -> pyrimidine nucleotide synthesis
  • PRPP + base -> 1 step (salvage) -> UMP -> pyrimidine nucleotide synthesis
160
Q

dNMP’s, dNDP’s, and dNTPs

A
  • deoxynucleotides

- loses OH group from pentose sugar

161
Q

origin of ring atoms in purines

A
  • nitrogenase base that is built on PRPP comes from different precursor molecules
  • glycine, glutamine, aspartic acid, formate, CO2 -> contribute to different parts of the purine ring
162
Q

De Novo biosynthesis of purines

A
  • purines have 2 rings
  • rings are being built on the PRPP
  • ATP is used -> multienzyme energy consuming rxn
  • glutamine gives amine group to 1’ of PRPP (rate limiting step) -> forms 5-phosphate-beta-D-ribosylamine (denoted at “R”) -> this is the ribose sugar that the base will now be built off of
  • glycine contributes to the ring -> forms glycinamide ribonucleotide (GAR)
  • attachment of carbon from tetrahydrofolate -> forms formylglycinamide ribonucleotide (FGAR)
  • glutamine gives another amine group -> formylglycinamide ribonucleotide (FGAM)
  • first ring of purine is formed -> 5-aminoimidazole ribonucleotide (AIR)
  • carbon is added from CO2 (bicarbonate)
  • aspartate add another nitrogen
  • fumarate adds
  • tetrahydrofolate add more carbon
  • water molecule is released -> closes up the second ring
  • Inosinate (IMP) -> final precursor for all purine synthesis -> can be built into different purines
  • base is built onto the ribose sugar
163
Q

synthesis of AMP and GMP from IMP

A
  • inosinate (IMP)
  • adenylate (AMP)- precursor of ATP (uses GTP to make)
  • guanylate (GMP)- precursor of GTP (uses ATP to make)
  • AMP:
  • amino group comes from aspartate
  • GMP:
  • amino group comes from glutamine
164
Q

synthesis of AMP from IMP

A
  • amino group comes from aspartate
  • inosinate (IMP)
  • adenylate (AMP)- precursor of ATP (uses GTP to make)
  • IMP -> aspartate add amino group -> uses GTP -> adenylosuccinate intermediate -> fumarate -> adenylate (AMP)
165
Q

synthesis of GMP from IMP

A
  • amino group comes from glutamine
  • guanylate (GMP)- precursor of GTP (uses ATP to make)
  • inosinate (IMP)
  • IMP -> NAD+ accepts H -> xanthylate (XMP) intermediate -> amino group comes from glutamine -> uses ATP -> guanylate (GMP)
166
Q

summary of De Novo biosynthesis of purine

A
  • de novo biosynthetic pathway builds the base portion onto PRPP from available building blocks (Gln, Gly, and Asp)
  • 11 enzymatic steps
  • highly conserved throughout life (bacteria and humans -> not all parasites tho)
  • energy intensive
  • uses several amino acids (Gln, Gly, Asp) as building blocks
  • requires cofactors
  • 11 enzymes in most prokaryote
  • 6 enzymes in humans (several bi-functional and tri-functional enzymes -> combined)
  • first intermediate with a full purine ring is inosinate (IMP)
  • ATP is used to phosphorylate GMP precursor, while GTP is used to phosphorylate AMP precursor
167
Q

salvage pathway of purine biosynthesis

A
  • level of purine nucleotides is maintained by 2 complementary pathways
  • salvage pathways makes purine nucleotides from PRPP and preformed bases
  • single enzyme step
  • maintains purine levels under ‘normal’ cellular conditions
  • you dont have to build nitrogenous base on top of the PRPP molecule (like in de novo) -> takes PRPP and hypoxanthine (recycled precursor of nitrogenous base) -> enzyme (hypoxanthine phophoribosyltransferase (HPRT)) combines them -> makes inosine monophosphate (IMP)
168
Q

regulation of purine biosynthesis in E. coli largely consists of feedback inhibition

A
  • PRPP synthesis is inhibited by ADP and GDP
  • glutamine-PRPP amidotransferase is inhibited by end-products IMP, AMP, and GMP
  • ADP can inhibit the rate limiting step (glutamine gives amine group to 1’ of PRPP) -> inhibits the whole rxn by inhibiting PRPP formation -> allosterically inhibits bc its the final product of the whole rxn
  • excess GMP inhibits formation of xanthylate from inosinate by IMP dehydrogenase
  • GMP and AMP concentration inhibit phosphorylation steps
169
Q

catabolism of purine

A
  • dephosphorylation (via 5’-nucleotidase
  • removal of pentose sugar (via nucleosidase) -> at this point (sugar and base are removed), purine bases can be salvaged
  • deamination and hydrolysis of ribose lead to production of xanthine
  • hypoxanthine and xanthine are then oxidized into uric acid by xanthine oxidase
  • uric acid -> urea is a waste
  • uric acid can undergo further oxidation:
  • primates: excrete much more nitrogen as urea via the urea cycle
  • plants and microorganisms: uric acid -> allantoin, urea or ammonia
  • fish: excrete much more nitrogen as ammonia than urea
  • spider and other arachnids lack xanthine oxidase
170
Q

summary of purine catabolism

A
  • the end products of purine catabolism depend upon the organism
  • primates, birds, reptiles and insects the end-product is uric acid
  • uric acid can be further catabolized by plants and microorganisms to make use of the nitrogen
  • final products can be allantoin, urea, or ammonia
171
Q

excess uric acid seen in gout (hyperuricemia)

A
  • painful joints (often in toes) due to deposits of sodium urate crystals
  • primarily affects males
  • crystal like -> grinding -> pain
  • may involve genetic under-excretion of urate and/or may involve overconsumption of fructose
  • treated with avoidance of purine-rich foods (seafood, liver) or avoidance of fructose
  • also treated with xanthine oxidase inhibitor allopurinol
172
Q

Lesch-Nyhan syndrome

A
  • x-linked mutations
  • HPRT (hypoxanthine phophoribosyltransferase) deficiency
  • results in neurological impairment, retarded motor development, involuntary movements and self-injurious behavior
  • build up of uric acid -> gout
  • improper purine salvage leads to hyperuricemia (gout)
  • bite lips and fingers
173
Q

allopurinol inhibits xanthine oxidase

A
  • blocks xanthine oxidase
  • used to treat gout
  • mimics hypoxanthine
  • allopurinol is converted to oxypurinol by xanthine oxidase
  • oxypurinol is a strong competitive inhibitor of xanthine oxidase
  • prevents formation of uric acid from hypoxanthine
  • reduce the amount of uric acid generation preventing crystallization in joints and gout
174
Q

recap of nucleotides and purine metabolism

A
  • nucleosides= nitrogenous base (purines or pyrimidines) + pentose sugar
  • nucleotides= nucleosides + phosphate
  • nucleic acids are polymers of nucleotides
  • both purine and pyrimidine biosynthesis can occur via salvage or de novo biosynthetic routes
  • the salvage pathway takes a preformed base and add it to a ribose sugar in a single enzymatic step
  • de novo purine biosynthesis is carried out in 11 highly conserved steps (11 enzymes in most prokaryotes, 6 in humans)
  • the final intermediate is IMP
  • de novo biosynthetic pathway is energy intensive (5 ATP) requires several amino acids and uses a folate cofactor at 2 steps
  • it is triggered during high nucleotide demand
  • degradation product of purine is uric acid (can be further oxidized into urea and ammonia)
  • excessive accumulation of uric acid causes hyperuricemia (gout)
175
Q

pyrimidine

A
  • single ring nitrogenous bases
  • carbon and nitrogen
  • cytosine
  • thymine (DNA) -> has a methyl group
  • uracil (RNA)
176
Q

pyrimidine nucleotide synthesis

A
  • PRPP + base -> 1 step salvage -> UMP
  • PRPP -> 6 step de novo -> UMP
  • UMP -> UDP -> UTP -> CTP -> CDP -> dCDP -> dCTP
  • NB: dNMP. dNDPs and dNTPs are deoxynucleotides
177
Q

5-phosphoribosyl-1-pyrophosphate (PRPP) is the precursor for the pentose sugar

A
  • for purine and pyrimidine
  • synthesized from ribose 5-phosphate of pentose phosphate pathway via ribose phosphate pyrophosphokinase
  • pyrophosphokinase makes this pentose sugar
  • precursor that is added to bases (purine or pyrimidine)
  • commonality in biosynthesis
178
Q

de novo synthesis of pyrimidine nucleotides

A
  • 1st step is generation of carbamoyl phosphate in the cytoplasm by CPSII (unlike in urea cycle CPSI makes carbamoyl phosphate in mitochondria)
  • CPS 2 gets nitrogen from glutamine
  • 3 active sits in CPSII
  • 1 active site reacts with bicarbonate
  • another reacts with ATP and hydrolysis
  • tunnel like structure in CPSII -> protects intermediates
  • glutamine gives up amine group
  • Gln + 2 ATP + HCO3- (bicarbonate) + H2O -> carbamoyl phosphate + Glu + 2 ADP + 2 Pi
  • precursor for the de novo synthesis for pyrimidine
  • unlike purine synthesis where PRPP is the precursor and bases are built on the sugar -> in pyrimidine synthesis the sugar gets attached to the bases later
179
Q

de novo synthesis of pyrimidine: building of ring structure

A
  • unlike purine synthesis, pyrimidine synthesis makes pyrimidine ring ( in form of orotate) and attaches it to ribose 5-phosphate
  • starts with ring synthesis
  • aspartate and carbamoyl phosphate provide the atom for the ring structure
  • aspartate transcarbamoylase (ATCase) is key enzyme that initiates this process -> combines aspartate with carbamoyl -> first precursor
  • orotate is the 1st pyrimidine base intermediate precursor
    1. carbamoyl phosphate combines with aspartate via aspartate trans-carbamoylase (ATCase) -> N-carbamoylaspartate -> RATE limiting step
    2. eventually forms orotate
  • nitrogenous base precursor is called orotate (IMP was precursor for purine)
    3. orotate is attached to ribose 5-phosphate sugar
    4. base undergoes decarboxylation -> forms uridylate (UMP) -> the first pyrimidine base and precursor for other pyrimidines!
    5. UMP gets converted to UTP (uridine-5-triphosphate) via phosphorylation
    6. UTP converts to CTP (cytidine 5’-triphosphate) via amination using CTP synthase -> amine gets attached from glutamine
180
Q

orotate

A

-orotate is the 1st pyrimidine base intermediate precursor
-derives most of its atoms from aspartate (amino acid that add with carbamoyl phosphate)
-some atoms are from glutamine and bicarbonate (bc carbamoyl phosphate is made from glutamine and HCO-3)
(-most of the atoms of purine come from glycine)

181
Q

regulation of pyrimidine biosynthesis is also via feedback inhibition

A
  • think back: aspartate transcarbamoylase (ATCase) attaches carbamoyl phosphate -> forms carbamoyl aspartate -> RATE LIMITING STEP
  • aspartate transcarbamoylase (ATCase) is inhibited by end-product CTP and is accelerated by ATP
  • allosteric
  • prevents the biosynthesis of biosynthesis
  • excess CTP inhibits
182
Q

salvage biosynthesis of pyrimidine

A
  • both purines and pyrimidines can be made de novo or using salvage/recycling pathways
  • uracil phosphoribosyltransferase (UPRT) combines/couples uracil (nitrogenous base) to PRPP (sugar) to make uridine (UMP)
  • UMP is first precursor
  • UMP can then be converted to UTC and CTP
  • uracil is the breakdown product of UTP and what is being recycled
183
Q

DNA vs. RNA

A
  • DNA has deoxysugars
  • DNA uses thymine and RNA uses uracil
  • 2’ carbon has H in DNA
  • 2’ carbon in RNA has OH
  • DNA is double stranded
  • RNA is single stranded
  • DNA: thymine, cytosine, adenine, guanine
  • RNA: uracil, cytosine, adenine, guanine
184
Q

ribonucleotides are precursors of deoxyribonucleotides (dNTPs)

A
  • same for purine and pyrimidine
  • deoxyribonucleotides are made by reduction of the corresponding ribonucleotide and are not made de novo
  • *deoxyribonucleotides are always synthesized from the ribonucleotides (ribose comes first)
  • ribonucleotides have the ribose sugar (2’ OH group)
  • deoxyribose sugar has 2’ H
  • conversion of ribose to deoxyribose is catalyzed by ribonucleotide reductase (RNR)
  • ribonucleotide is the precursor
  • pyrimidine:
  • UMP -> UDP -> dUDP -> dUTP -> dUMP -> dTMP -> dTDP -> dTTP
  • UMP -> UDP -> UTP -> CTP -> CDP -> dCDP -> dCTP
185
Q

ribonucleotide reductase

A
  • uses 2 H that come from NADPH (reduce the substrate) and carried by protein thioredoxin or glutaredoxin
  • NADPH also serves as the electron donor
  • funneled/carried through glutathione or thioredoxin pathways
  • this enzyme uses a free radical in the mechanism
  • makes use of cysteine (sulfur)
  • glutaredoxin is in oxidized form when there are disulfide bridges and reduced when there are not
  • regulated allosterically
  • regulated by substrate (ribonucleotides) and products (deoxyribonucleotide) -> keeps a constant pool of nucleotides
  • minimizes error
186
Q

structure of ribonucleotide reductase

A
  • tetramer
  • 2 alpha subunits
  • 2 beta subunits
  • active site has cysteine molecules
  • theoretical generating amino acid that gives up its proton and accepts etc.
  • many binding sites for regulation
  • huge amount of allosteric regulation
187
Q

proposed mechanism for ribonucleotide reductase

A
  • uses a free radical in the mechanism
  • generated at an iron center
  • uses NADPH as a proton donor
  • regulated allosterically
  • free radical active group strips off a proton from the ribonucleotide -> generates free radical-
  • H from the cysteine molecule will bind at 2’ OH group -> forms metastable molecule
  • water comes out from 2’
    • charge on 2’ -> another H from cysteine comes and bind to 2’ -> converts to deoxyribose sugar
  • H returned back to 3’
  • probable theory
188
Q

regulation of ribonucleotide reductase by dNTPs

A
  • highly controlled by substrate and product (and ATP)
  • tight control over dNTP ratio is essential
  • deficiency in any dNTP is lethal -> cant replicate DNA
  • excess of any one dNTP is mutagenic -> increased probability of incorporating the wrong dNTP
  • RNR also alters its oligomeric state based on overall ATP levels
  • allosterically regulated
  • substrate specificity site: allosteric regulation- regulates substrate binding and type of substrate that binds
  • primary regulatory site: allosteric regulation- regulates overall activity of enzyme (speed up or slow down, or completely inhibit) -> product or ATP can bind
  • excessive amount of ATP regulates what substrates can bind
  • keeps a constant pool of nucleotides
  • A pocket-> when ADP or ATP is bound at the allosteric site, the enzyme accepts UDP and CDP at the catalytic/active site
  • B pocket -> if dGTP is bound at substrate specificity site, then ADP binds at active site
  • C pocket -> dTTP, then GDP binds at active site
189
Q

thymidine deoxyribonucleotides (dTTPs)

A
  • in all the other cases, dNTPs are made by reduction of the corresponding nucleotide
  • there is no TMP, TDP, or TTP made (no thymine in RNA)
  • ribose version of thymidine nucleotide is not made
190
Q

1,3-bisphosphoglycerate

A
  • 1-phospho -> phosphoanhydride (high energy)

- 3-phospho is phosphoester (low energy)

191
Q

pyrimidine catabolism

A
  • requires a nucleotidase and nucleosidase

- end product is malonyl-CoA

192
Q

N5,N10-methylene THF

A
  • cofactor
  • used by thymidylate synthase to make dTMP
  • serine donates a hydroxymethyl group to THF (via SHMT enzyme)

Biochemistry (8 decks)

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