Lecture 4 Flashcards
(47 cards)
1
Q
Essential Requirements
A
- Two broad classes of metabolism
– Catabolic – energy releasing, used to
drive non-spontaneous reactions
– Anabolic – energy consuming,
biosynthesis of macromolecules - Many elements essential for microbial metabolism
- All require carbon source
– Essential for synthesis of overwhelming majority of biological macromolecules (proteins,
nucleic acids, lipids, etc.)
– Broad spectrum of potential sources (amino acids, fatty acids, organic acids, sugars,
nitrogen bases, aromatic compounds, etc.)
2
Q
Essential Requirements cont
A
- All require nitrogen source
– Essential for synthesis of many biological macromolecules (amino acids, nucleotides)
– NH4
+ preferred source for most organisms
– Not readily available form many environments (soil)
– Many capable of using NO3
-
– Few capable of using N2
, nitrogen fixing organisms - All require phosphorous source, essential for production of lipids and nucleotides, organic
and inorganic phosphates suitable sources
3
Q
essential requirements cont 2
A
- All require sulfur source, essential for synthesis of some amino acids, SO4
2- and HSsuitable
sources - All require K+ essential element, required for activity of multiple enzymes
- All require Mg2+ essential for activity of numerous DNA binding proteins and enzymes
- All organisms require Iron, essential cofactor for numerous enzymes
- Produce siderophores to facilitate uptake from environmental sources (Fe2+ under anoxic
conditions, Fe3+ under oxic conditions) - Different organisms use different siderophores
4
Q
essential requirements cont 3
A
- Hydroxamate (siderophore) produced and secreted from cell
- Binds environmental iron source with high affinity
- Hydroxamate/Iron complex bind receptor on plasma
membrane - Hydroxamate/Iron complex transported into cell
- Iron removed from hydroxamate, used by cellular enzymes
- Hydroxamate secreted to repeat cycle
5
Q
Specific Requirements for Some Organisms
A
- Some organisms require Ca2+ for cell wall stability
- Some halophilic organisms require Na+
- Some organisms require trace metals, used as cofactors (B, Cr, Co, Cu,
Mo, Ni, Se, W) - Some organisms require growth factors, organic molecules essential for
viability (vitamins, amino acids, nitrogen bases)
6
Q
Transfer of Electrons - Redox Reactions
A
- Oxidation/Reduction reaction
- Involves transfer of electrons and their energy
- Actually 3 reactions
- By losing electrons Fe is oxidized – electron donor
- By gaining electrons O is reduced – electron acceptor
- Can involve proton transfer as well
Helpful hint: Atom is reduced if charge is reduced
7
Q
Redox Reactions with NAD+/NADH
A
- Involves shuttling through reduced (NADH) and oxidized forms (NAD+
) - Enzyme binds NAD+ and electron donor
- Catalyzes transfer of hydrogen atom (and its electrons) from electron donor
- Net result is reduction of NAD+
to NADH - Energy from electron donor now transferred to NADH
8
Q
Redox Reactions with NAD+/NADH cont
A
- Second enzyme binds NADH and second substrate
- Hydrogen atom (and its electrons) transferred to second substrate
- NADH oxidized to NAD+
, second substrate now reduced - Each half reaction spontaneous, direct reaction often is not
- Redox reactions make non-spontaneous reactions possible by coupling two spontaneous
reactions
9
Q
Energy Storage
A
- Redox reactions often produce energy
- Energy produced not always needed at time
- Needs means to store energy for later use
- Often stored in phosphorylated compounds
- Examples include ATP, PEP, Glucose 6-phosphate
- Energy “stored” in anhydride bonds and ester bonds
- Energy released upon hydrolysis of bonds
- Hydrolysis of anhydride bonds releases much more energy than ester bonds
10
Q
Thioesters as Energy Storage
A
- Carboxylic acid – sulfur bonds (thioester) also energy rich
- Liberate energy when bond breaks
- Used for synthesis of ATP
- Best example – acetyl Coenzyme A
- acetyl-S-CoA + H2O + ADP + Pi → acetate- + HS-CoA + ATP + H+
11
Q
Long-Term Energy Storage
A
- ATP currency for energy, not suitable of storage of excess energy
- Complex polymers used for long term storage
- Glycogen for bacteria and animals, starch for plants
- Many bacteria use other polymers for storage – poly-b-hydroxybutyrate
- Sulfur chemolithotrophs use elemental sulfur
- All can be oxidized to produce ATP
12
Q
Bioenergetics
A
- Actual thermodynamic principles beyond scope of course (I spend entire lectures on this for
other courses) - Need only know that endergonic reactions consume energy and are non-spontaneous
- Exergonic reactions release energy and are spontaneous
- Exergonic reactions does not necessarily indicate fast reaction rate
- May proceed too slow to support biological processes
- Due to energy of activation, energy required to break existing bonds to allow new ones to
form
13
Q
Enzymes Decrease Activation Energy
A
- Biological processes in the absence of enzymes can require much energy to start reaction
- Can force reactions to proceed at very slow rate
- Enzymes decrease energy required to start
chemical reactions - Allows reaction to proceed at biologically relevant
rate
14
Q
Enzyme Catalysis
A
- Enzyme has active site, complementary shape to substrate
- Allows binding of substrate, active site contains reactive group
- Reactive group stresses chemical bonds that need to be broken
- Decreases the energy required to start the reaction
15
Q
Requirements for Enzyme Activity
A
- Many enzymes require additional factors, two categories
– Prosthetic groups – additional molecule covalently attached to enzyme (eg. Heme group
present in cytochromes)
– Coenzymes – non-covalently bound, capable of dissociation, binding affinity may be
extremely high - Coenzymes often derivatives of vitamins
- NAD+ and NADH derivatives of niacin
- Essential for catalysis of redox reactions by many enzymes
16
Q
Generation of ATP
A
- Sugars stored in starch and glycogen used to produce ATP
- Metabolism used depends on availability of oxygen
- Anaerobic environments require fermentation
- Transfer of phosphates through redox reactions
- No terminal electron acceptor required, small amount of ATP produced
- Availability of terminal electron acceptors allows respiration
- Possible under aerobic and anaerobic conditions
- Differ in terminal electron acceptor
- Relies on generation of proton motive force, increases ATP produced
17
Q
Glycolysis – a Type of Fermentation – Stage I
A
- Requires 3 stages of reactions, Stage I begins with phosphorylation of glucose to generate
glucose 6-phosphate - Catalyzed by hexokinase, consumes 1 ATP
- Isomerase converts to fructose 6-phosphate
- Phosphofructokinase attaches second phosphate group, consumes 1 ATP
- Fructose 1,6-diphosphate shunted into stage II reaction
18
Q
Glycolysis–a Type of Fermentation–Stage II
A
- Aldolase converts fructose 1,6-diphosphate to 2 molecules of glyceraldehyde 3-phosphate
- Glyceraldehyde 3-phosphate dehydrogenase converts both to 1,3-bisphosphoglycerate in
presence of inorganic phosphate
– Results in transfer of electrons to NAD+
, reduced to NADH
– Transfer of electrons makes reaction spontaneous - Phosphoglycerokinase transfers phosphate from 1,3 bisphosphoglycerate to ADP, generates
2 molecules of ATP, one for each 1,3 bisphosphoglycerate
19
Q
Glycolysis–a Type of Fermentation–Stage II cont
A
- Removal of phosphate generates 2 molecules of 3-phosphoglycerate
- Phosphate transferred to 2’ carbon by phosphoglyceromutase, converts to 2-
phosphoglycerate - Enolase converts 2-phosphoglycerate to phosphoenolpyruvate (PEP)
- Pyruvate kinase transfers phosphate from PEP to ADP, converts PEP to pyruvate
- Generates 2 molecules of ATP, one for each PEP
20
Q
Glycolysis–a Type of Fermentation–Stage III
A
- Glycolysis requires NAD+
, reduced to NADH when glyceraldehyde 3-P is converted to 1,3-
bisphosphoglycerate - NADH must be oxidized to replenish supply of NAD+
, accomplished by Stage III reactions - Some bacteria transfer protons (and electrons associated with them) from NADH to
pyruvate, converts to lactate, catalyzed by lactate dehydrogenase - Net result, oxidation of NADH to
NAD+, replenishes supply required
for Stage II reactions
21
Q
Glycolysis–a Type of Fermentation–Stage III cont
A
- Yeast and some bacteria use pyruvate decarboxylase to convert pyruvate to acetaldehyde
- Alcohol dehydrogenase transfers protons from NADH to acetaldehyde, generates ethanol
- Net result, oxidation of NADH to NAD+
, replenishes supply required for Stage II reactions - Glycolysis results
in net yield of 2
ATP
22
Q
Maximizing ATP Production – Respiration
A
- Glycolysis only generates 2 ATP
- Respiration generates 36 ATP
- Relies on generation of proton motive force
- Requires generation of “energized” membrane
- Protons accumulate on one side of membrane
- Hydroxyl groups accumulate on opposite side
- Discharge of proton motive force drives production of ATP
- Process referred to as oxidative phosphorylation
23
Q
The Citric Acid Cycle – Fueling Electron Transport
A
- Only need to know that 4 NADH generated
- Donates protons/electrons to electron
transport chain through complex I - Each generates 3 ATP through oxidative
phosphorylation, net yield 12 ATP - Generates 1 FADH2
- Donates proton/electron to electron
transport chain through complex II - Generates 2 ATP through oxidative
phosphorylation, net yield 2 ATP
24
Q
The Citric Acid Cycle – Fueling Electron Transport
A
- One GTP generated, produces one ATP
- Two molecules pyruvate generated by
glycolysis - Allows for two revolutions of the TCA cycle
- Glycolysis contributes 2 NADH, allows
production of 6 ATP - Glycolysis contributes production of 2 ATP
- Total net yield – 38 ATP
25
Generating the Proton Motive Force
* Involves transfer of electrons from molecules with
decreasing reductive potential
* Flavoproteins bound to flavine mononucleotide
* Accept 2 protons and two electrons from NADH, only
donate electrons
* Electrons donated to iron sulfur proteins, either Fe2
S2
or Fe4
S4
, iron molecules coordinated by Cys residues
* Iron sulfur proteins transfer electrons to proteins
bound to quinones
26
Generating the Proton Motive Force cont
* Quinones transfer electrons to cytochrome proteins
* Cytochrome proteins transfer electrons to other cytochromes
through other iron sulfur proteins
* Protons transferred to one side of membrane at multiple steps
* Net result, accumulation of protons on one side of the membrane
* Proton motive force now generated
27
Putting it All Together
* NADH transfers protons and electrons to FMN complex – complex I
* FMN transfer protons to one side of membrane, transfer electrons to iron sulfur proteins,
part of complex I
* Iron sulfur proteins transfer electrons to quinones, reduces to QH2
* Protons transferred to one side of membrane as electrons transferred from quinolone to
cytochrome B
* Cytochrome B transfers electrons to
cytochrome C1
through iron sulfur
protein
* Cytochrome b and iron sulfur
proteins comprise complex III
28
Putting it All Together cont
* Cytochrome c1
transfers electrons to cytochrome, cytochrome c transfers electrons to
cytochrome a
* Cytochrome a transfers electrons to cytochrome a3
* Transfer of electrons to cytochrome a3
to oxygen generates water and transfers protons to
one side of membrane
* Cytochrome a/a3
comprise complex
IV
29
Using the Proton Motive Force to Make ATP
* Complex II further contributes to generation of proton motive force
* Bypasses complex I, allows FADH2
to transfer 2 protons and 2 electrons to quinolones
* Protons transferred to one side of membrane
* Electrons continue down electron transport chain
* Collectively responsible for
generation of proton motive force
30
Using the Proton Motive Force to Make ATP cont
* Requires ATP synthetase (Complex V), 2 domains – F0 (a, b2
, c12) and F1
(a3
, b3
, g, e, d)
* Domain F0
spans plasma membrane, 3 proteins
– a protein forms channel, allows protons to flow down concentration gradient
– Proton flow through a induces rotation of c proteins
* Rotation of c proteins induces torque on F1
– Due to interaction with eg subunits
– Causes rotation of eg subunits
31
Using the Proton Motive Force to Make ATP cont 2
* Rotation of eg subunits induces conformational change in b subunits
* Allows binding of ADP and Pi
* b subunits return to original conformation, generates ATP from ADP and Pi bound
* Process referred to as oxidative phosphorylation
32
Convergence of Glycolysis and Respiration
* Pyruvate an intermediate product in glycolysis
* Reduced to fermentation products
* Replenishes NAD+
required for ATP production
* Possible to increase amount of ATP produced
* Pyruvate can enter into the Citric Acid Cycle
* Results in complete oxidation to CO2
*NADH oxidized to NAD+ by donating proton and electron to electron transport chain
* Still generate ATP from glycolysis, gain additional ATP from oxidative phosphorylation
33
Alternate Electron Acceptors – Anaerobic Respiration
* Many ecological environments limiting for oxygen
* Limits availability for respiration
* Many organisms use alternate terminal electron acceptors
* Include S0
, NO3
-
, SO4
-
, and organic electron acceptors
* Termed anaerobic respiration or chemoorganotrophy
* Generates less energy, compounds have lower affinity for electrons
34
Alternate Electron Acceptors – Anaerobic Respiration cont
* E. coli facultative anaerobe, uses oxidative phosphorylation for energy metabolism if oxygen
present
* Uses anaerobic respiration if oxygen absent, initiates with similar mechanism
* Proton donors from glycolysis and Krebs cycle donate protons and electrons associated with
them to FMN in Complex I
* Electrons transferred to Fe/S to quinone to
nitrate reductase, nitrate used as terminal
electron acceptor
* Process also initiates with Complex II
* Does not use Complex IV, fewer protons moved,
less ATP produced, slower growth
35
Alternate Electron Donors – Chemolithotrophy
* Many organisms use inorganic molecules as electron donors
* Include H2
S, H2
, Fe2+, and NH3
* Still require terminal electron acceptor
* May be anaerobic or aerobic metabolism
* Type of terminal electron acceptor defines aerobic vs. anaerobic metabolism
36
Alternate Electron Donors – Chemolithotrophy
* Ralstonia eutropha initiates electron transport with H2
, donates protons and electrons to
membrane bound hydrogenase
* Protons moved to periplasm, electrons transferred to quinone
* Quinone transfers electrons to cytochrome bc1
complex (analogous to Complex III), 4 more
protons moved to periplasm
* Electrons sequentially transferred to cytC and
then terminal reductase (analogous to Complex
IV)
* Electrons transferred to oxygen, aerobic
respiration in this example
37
Alternate Means of Generation of Proton
Motive Force – Phototrophy
* Many organisms undergo photosynthesis – use energy from light to generate proton motive
force
* All use ATP generated as energy for biosynthetic reactions
* Some use organic compounds for biosynthetic reactions – photoheterotrophy
* Some use CO2
for biosynthetic reactions – photoautotrophy
38
Alternate Means of Generation of Proton
Motive Force – Phototrophy cont
* Light harvesting centers (LHII and LHI) absorb energy from photons in light, uses
bacteriochlorphyll P870
* Energy absorbed excites to P870*
, electron released and donated to bacteriopheophytin
(Bph)
* Bacteriopheophytin transfers electrons sequentially to quinone then cytochrome bc1
complex (analogous to Complex III)
* Cyt b transfers electrons to cyt c1
then cyt c2
then back to
light harvesting center
* Protons moved to periplasm by cytochrome bc1
complex,
proton motive force discharged through ATP synthase
39
Carbon Fixation – Generation of Sugars
* Photoautotrophic bacteria and plants use CO2
to produce sugars, CO2 used with ATP and NADPH in the CALVIN
CYCLE to produce sugars
* Cycle starts with addition of carbon atom from 6 CO2
to 6 molecules of ribulose 1,5-bisphosphate – CARBON
FIXATION
* Catalyzed by RUBISCO – ribulose bisphosphate
carboxylase/oxygenase
* 6 carbon intermediate molecule broken down into
two molecules of 3-phosphoglycerate, total yield of
12 molecules of 3 phosphoglycerate
40
The Calvin Cycle – Generation of Sugars
* Second stage involves reduction of intermediates to produce carbohydrates
* 12 ATP from light reactions used with to convert 12 molecules of 3-phosphoglycerate to 12 molecules of
1,3-bisphosphoglycerate
* 12 NADPH from light reactions used to reduce 12
molecules of 1,3-bisphosphoglycerate to 12
molecules of glyceraldehyde 3-phosphate –
reduction reaction
* Two molecules of glyceraldehyde 3-phosphate
exported to cytoplasm, combined to create hexoses
(glucose, fructose)
41
The Calvin Cycle – Generation of Sugars cont
* Third stage involves regeneration of ribulose bisphosphate (RuBP)
* 10 molecules of glyceraldehyde 3-phosphate remain in stroma
* Carbon atoms combined and rearranged to generate 6 molecules of ribulose monophosphate
* 6 molecules of ATP from light reactions used to
convert 6 molecules of ribulose monophosphate to
6 molecules of ribulose 1,5-bisphosphate,
completes Calvin cycle
42
Nitrogen Fixation
* Amino acids and nucleotides require nitrogen for biosynthesis, most organisms acquire from
organic source
* Some organisms initiate the nitrogen cycle by producing NH3
from N2 – NITROGEN FIXATION
* NH3
readily incorporated into macromolecules
* N2 has triple covalent bond, difficult to reduce, requires rapid successive reduction
* Catalyzed by NITROGENASE, dimer of dinitrogenase and dinitrogenase reductase
* Electron transfer is rapid, no intermediates produced
* Facilitates reduction of N2 to NH3
* Nitrogenase poisoned by O2
, cyanobacteria create anoxic environment in heterocysts, other
organisms use oxygen based metabolism to create anoxic microenvironment in cytoplasm
43
Nitrogen Fixation cont
* Electron donors often derived from end stage of glycolysis, 4 pyruvate combine with 4 CoA
* 8 electrons released, accepted by electron donor (4 flavodoxin or 4 ferredoxin), electron
donor reduced
* 8 electrons transferred to 4 dinitrogenase reductase, becomes reduced
* Association with ATP followed by hydrolysis lowers reduction potential of dinitrogenase
reductase, allows transfer of electrons to dinitrogenase
* 2 molecules of ATP required per electron, 16 ATP total
* 4 reduced dinitrogenase produced, 6 electrons transferred to N2
* Produces 2 NH3
, remaining two electrons accepted by 2H+
* Combine to produce H2
44
Biosynthesis of Polysaccharides
* Simple sugars may be in abundance during growth under certain conditions
* Excess monomers stored as polymers, commonly glycogen for many bacteria
* Glucose converted to activated form, commonly uridine diphosphoglucose (UDPG), may be
adenosine diphosphglucose (ADPG)
* Activated sugar added to growing glycogen polymer via a-1-4 linkage
* Branches made by a-1-6 linkage, addition continues until excess sugar exhausted
* Activate sugars also used in synthesis of peptidoglycan
45
Synthesis of Monosaccharides
* Organism may be growing in absence of simple sugar, possible to synthesize glucose –
GLUCONEOGENEISIS
* If phosphoenolpyruvate available, glycolysis reversed to produce glucose
* If phosphoenolpyruvate not available, possible to shunt carbon compounds of varying length
through the Krebs cycle
* Oxaloacetate intermediate from Krebs cycle converted to phosphoenolpyruvate, CO2
liberated
* Glycolysis reversed using phosphoenol pyruvate produced
* Glucose synthesized
46
Synthesis of Monosaccharides
cont
* Previous slide delineates synthesis of hexoses, pentoses also essential for viability
* Process starts with glucose-6-phosphate, carbon removed as CO2
, produces ribulose-5-
phosphate (ketose)
* Isomerized to ribose 5 phosphate (aldose), used for nucleotide biosynthesis
* Ribose-5-phosphate circularized, nitrogenous base added to 1’ carbon to produce
ribonucleotide, used for RNA synthesis
* Ribonucleotides reduced by ribonucleotide reductase
using NADPH to remove 2’-OH to produce
deoxyribonucleotide, used for DNA synthesis
47
Synthesis of Amino Acid
* Bacteria may grow in the absence of exogenous protein, requires
synthesis of amino acids, many produced from intermediates
from glycolysis
* Pyruvate converted into alanine family of amino acids
* 3 – phosphoglycerate converted into serine family of proteins
* Phosphoenolpyruvate converted into chorismate, used for synthesis of aromatic amino
acids
* Many amino acids synthesized from intermediates from the Krebs cycle
* a-ketoglutarate used for synthesis of glutamate family of amino acids
* Oxaloacetate uses for synthesis of aspartate family of amino
acids
