BIOL 2421 Microbiology Lecture Notes: Microbial Metabolism Dr. Weis
Metabolism:
Def: sum of all biochemical reactions in living organisms
Requirements: Energy, enzymes
Two types of metabolic reactions:
a) catabolism: breakdown of complex chemical compounds, releasing energy
e.g. hydrolytic, exergonic
b) anabolism: synthesis of chemical compounds, requiring energy
e.g. dehydration synthesis, endergonic
Metabolism
reaction rates depend on the number of molecules (substrate) available and
the activation energy.
The rate limiting step is the slowest step in the chemical process.
Reaction rates can be affected by temperature (heat, cold), pH, pressure/concentration, distance.
Energy: the ability to do work or to move matter.
Forms: Kinetic and Potential
Energy use: Chemical, Electrical, Mechanical, Electromagnetic (radiation)
Chemical Energy Types: ATP, GTP, UTP
Synthesized from phosphate and a nitrogen base (Adenine, Guanine, Uracil)
ADP + Pi + E çè ATP
Recall that when energy is released to do the work of the cell, part of that energy is lost to the environment as heat.
Enzymes:
Normally protein based catalysts that lower the activation energy of the reaction.
The activation energy is the amount of energy needed to disrupt the stable
electron configurations of a molecule so that the electrons can be rearranged.
Enzymes:
a) Act on specific substances (substrate) to create a product
b) Catalyze one reaction
c) Contains an active functional site
d) Three dimensional shape (primary, secondary, tertiary, quaterinary)
e) Exist in both active and inactive forms
f) Are not changed or used up in the chemical process/reaction
g) Named for chemical class of reactions catalyzed, -ase ending
h) Two parts: apoenzyme (protein) + cofactor / coenzyme = holoenzyme
Examples of coenzymes (organic cofactors)
CoEnzyme A
Vitamin
Vitamin B based: NAD+, NADP+, FAD
Examples of cofactors (nonorganic molecules)
Metal Ions of Copper, Iron, Zinc, Magnesium, Calcium, Manganese
Categories of Enzymes
a) Hydrolases
add water to catabolize molecule
b) Isomerases
rearrange atoms within molecule
c) Ligases/Polymerases
join molecules together
d) Lyases
split molecules apart
e) Oxidoreductase
redox reactions
f) Transferases
move functional group between molecules
Factors affecting enzymes:
Temperature, pH, acids, bases, heavy metals, alcohol, UV light:
Changes beyond optimum can cause denaturing
The breakdown of the 3D structure.
Might be reversible as long as the enzyme was not coagulated
Factors affecting enzymes:
substrate concentration, if constituents high enough, can saturate enzyme’s active site causing no further increase in reaction rate.
inhibitors: competitive and noncompetitive ; reversible and irreversible
* Competative inhibitors fill active site and block enzyme, no Rxn
i. Reversible: can be competitively removed
ii. Irreversible: bind permanently
* Noncompetative inhibitors bind to another site called the allosteric site and changes the shape of the active site making it nonfunctional.
Allosteric inhibitors that bind can play a role in feedback inhibition for the end product.
i. Inhibitory: stops enzyme activity
ii. Excitatory: activates the inactive enzyme
Recent findings indicate another different
enzyme structure:
From ribosome, called ribozymes.
Functions
like a normal protein based enzyme, but only works on RNA
to remove sections
and splice sections of RNA.
This "new" enzyme
is now considered the primary core of a ribosome.
Summary:
Enzyme specificity
Induced Fit
Enzyme substrate complex
Concentration of enzyme determines metabolic rate.
Enzyme synthesis is regulated by DNA and based on negative feedback controls.
Energy
Production
Redox reactions
Oxidation is the loss/removal of electrons from an atom or molecule
Reduction is the gain of an electron
Redox processes are controlled series of coupled reactions to prevent large
amounts of heat from being released all at once.
Dehydrogenation
Use of hydrogen in oxidative reactions. The removal of the electron belonging to hydrogen and leaving a proton or H+ atom that may be released to the surrounding medium.
The energy released during Redox reactions in trapped in the chemical bonds that form ATP in a process called phosphorylation. ADP + E + Pi forms Adenosine-Pi ~Pi~Pi (ATP)
Electrons do not normally exist free in cytoplasm. If released from an atom, electrons will be carried on Vitamin derivatives
NAD+ --> NADH
NADP+ --> NADPH
FAD--> FADH2
Phosphorylation Types
A> Substrate level: High energy phosphate is directly transferred to ADP, seen in glycolysis steps.
B> Oxidative Phosphorylation: electrons are transferred to carriers down an electron transport chain (located in the cytoplasm of prokaryotes and the mitochondrial matrix of Eukaryotes) to the final electron acceptor, which could be O2 or some other molecule.
The release of energy from one electron carrier to the next to help make ATP is called chemiosmosis.
C> Photophosphorylation: converts light energy to chemical energy (ATP) in photosynthetic cells. This chemical energy is then used to synthesize organic molecules.
Glucose Metabolism
6 carbon carbohydrate oxidized as primary source of cellular energy (E).
Catabolism creates “waste” products
Microorganisms can use cellular respiration or fermentation to produce E.
STEP ONE: Glycolysis
Splitting or breakdown of glucose into 2 molecules of Pyruvate [Pyruvic acid]
If other carbohydrates are used, must be broken down into intermediary
Occurs in Cytoplasm
Anaerobic
Pathways : Embden-Meyerhoff, Pentose-Phosphate, Entner-Doudoroff
Classic Pathway (Embden-Meyerhoff)
End products:
2 Pyruvic Acid
2 net ATP [4 ATP made – 2 ATP used]
ATP produced by substrate level phosphorylation
Mg++ required as cofactor
2NADH
2H2O
Pentose - Phosphate Pathway
Breakdown of 5 and 6 carbon sugars
End Products:
1 ATP
1 NADPH
CO2
7, 6, 5, 4 Carbon sugar intermediates that can go on to
form AA
form nucleotides
enter glycolysis
perform photosynthesis
Entner-Duodoroff Pathway
Breakdown of glucose
Use different enzymes than glycolysis
End Products
1 net ATP [2ATP produced – 1 ATP used]
1 NADPH
1 NADH
2 Pyruvic acids
2 H2O
seen in some G(-) bacteria such as Pseudomonas
Seen in some G(+) bacteria such as Enterococcus faecalis
STEP TWO: Presence or absence of Oxygen
With oxygen: Aerobic respiration
Without oxygen:
Aerobic Respiration
Final electron acceptor is Oxygen (O2)
Krebs Cycle + Oxidative Phosphorylation
Redox reactions that oxidize pyruvic acid derivatives and reduce coenzymes such as NAD+ and FAD
Pyruvic acid--> CO2 [decarboxylation] + Acetate --> Acetate +CoA + NADH--> Acetyl CoA
Oxaloacetic acid + Acetyl CoA à Citric Acid (6-Carbon)
Krebs Cycle {TCA, Citric Acid} Summary:
6 Carbon [C] Citric acid goes to 5 Carbon alpha-ketoglutaric acid + NADH + CO2
5 C molecule goes to a 4 C + NADH + CO2 + ATP via substrate level phosphorylation
4 C molecule undergoes several changes; H comes off to be picked up by FAD and NAD+
Generate Oxaloacetic acid that combines with Acetyl CoA to form Citric Acid
Electron Transport Chain [ETC]
In the plasma membranes of prokaryotes
Contains three carrier molecules for redox reactions
a) Flavoprotiens based on Vitamin B
b) Cytochromes based on Iron pigments
c) Coenzyme Q
Function:
Transfer of high E electrons from NADH to chain flavoproteins.
The H+ is pumped away from the chain and put on the other side of the plasma membrane in the Periplasm of bacteria.
The electrons are then transported
Either to Iron – Sulfur proteins if NADH started
Or to CoQ if FADH2 transported the electrons
Then continues down the cytochromes [B, C, A]
The last cytochrome, A3
Then passes the electrons to oxygen which becomes negatively charged
O2- attracted to the H+ that has come through the ATP synthetase wheel pump due to concentration gradients.
An ADP molecule is then joined to a phosphate to form ATP when a H+ moves across.
The electrons on the O2 combine with the H+ to form water.
Regenerates FAD+ and NAD+ for reuse in Krebs Cycle
Carrier molecules in ETC are diverse
a) Differ between bacterial genuses
b) Altered based on environmental changes
c) Cytochrome A+ A3 = Cytochrome oxidase in some bacteria
Cytochrome oxidase + bacteria : Pseudomonas, Neisseria
Cytochrome oxidase – bacteria : E. coli,
Salmonella, Proteus
The mechanism of making ATP via oxidative phosphorylation using the electron chain is called chemiosmosis.
38 total for Prokaryotes, 36 total of Eukaryotes (some energy is lost when electrons are shuttled through the mitochondria. No such loss occurs in Prokaryotes)
Prokaryote Summary: Glucose + 6 O2 ===> 6 CO2 + 6 H2O + 38 ATP + Heat
Final Electron Acceptors
Aerobic Respiration
Oxygen
O2 - + 2H+ --> H20 + ATP
Anaerobic Respiration:
Final electron acceptor is something other than Oxygen
Example: Nitrate Ion (NO3 -) as electron acceptor can generate one of the following:
NO2- nitrite ion
N2O nitrous oxide
N2 nitrogen gas
Sulfate Ion (SO4=) to form H2S (hydrogen sulfide gas)
Carbonate Ion (CO3=) to form CH4 (methane gas)
Fermentation
Anaerobic
Partial oxidation
Does not use Krebs Cycle or Oxidative phosphorylation
Use organic intermediary molecules as final electron acceptor to create end products
Small amounts of ATP are generated by substrate level phosphorylation.
Primary function is to regenerate NADH to NAD+ for glycolysis. There are small amounts of ATP available, since most energy stored in product
Pyruvic acid is converted into another organic product depending on the organism.
a) Lactic Acid
b) Acetic Acid
c) Acetone
d) Butyric acid
e) CO2
f) H2
g) Alcohols: Isopropyl, Ethanol
h) Contaminants that can cause tissue damage and death [necrosis]
Identification of end products is useful in identifying organisms as well as for use in industry or commercial products.
Two major types of fermentation are:
Lactic Acid Fermentation (Pyruvic acid to Lactic Acid) seen in bacteria
Alcohol Fermentation (Pyruvic acid to Acetaldehyde to Ethanol) seen in yeasts
Catabolism via proteases and peptidases so that the Amino
acids can cross the membrane.
To use in an energy pathway, they must be converted to other substances that
can enter the glycolysis or Krebs Cycle.
These processes are
a) Deamination : removal of the NH3 group (then converted to NH4+)
remaining molecule can enter an energy pathway
b) Decarboxylation: removal of the –COOH (carboxyl) group
d) Dehydrogenation: removal of the Hydrogen
Catabolism of the fats involve the splitting of the glycerol and fatty acids by lipases.
Glycerol can enter the glyolytic pathway
Fatty acids are converted to Acetyl CoA via Beta oxidation
Biochemical Testing for Bacterial Identifcation
Biochemical tests are designed to use metabolic processes to help:
Recall that nutritional classifications of bacteria categorize them as chemotheterotrophs
Metabolic Pathways for Energy Use
Energy is produced by aerobic respiration, anaerobic respiration, and fermentation.
45% of the energy generated is given off as heat.
Microbes
use the remaining ATP energy for life processes: metabolism, growth, responsiveness
and reproduction.
Specific examples include
a) active membrane transport
b) motility
c) generation of new cellular components via biosynthesis
Biosynthesis
A) Polysaccharide
Synthesize simple sugars from
Carbon intermediaries in the Krebs cycle
Amino Acids
Lipids (glycerol)
Stored as glycogen (Glucose-6 + ATP -> ADGP)
Cell wall components such as peptidoglycan (Fructose-6 + UTP -> UDPG)
B) Lipid
Glycerol + FA
Makes components of cell membrane
Makes components of cell wall and waxes [mycolic acid] in acid fast bacteria
Pigment production
Energy Storage
C) AA and Protein
Some microbes require a few essential amino acids, some can synthesize all
Glucose (or metabolism intermediaries) + inorganic salts with enzymes
Krebs cycle precursors such as pyruvic acid + amine -> Amino Acid
Processes using NH3
Amination
Transamination with Vitamin B6 as a coenzyme
AA form basis for proteins used as
Enzymes
Structural components
Toxins
Ribosomal ribozymes involved in protein synthesis
D) Purine and Pyrimadine
Nitrogen-bases. Purines are Adenine and Guanine. Pyrimadines are T,C,U
5 carbon sugar (pentoses) from alternate carbohydrate metabolism
AA [glutamine, aspartic acid] from Krebs cycle intermediaries, with carbon form N base ring
Nucleotides form DNA, RNA, ATP, NAD+, NADP
Phosphate group from ATP
Integration
of Metabolism
Share common intermediaries: Breakdown of one is used in synthesis
of another
Function in both catabolism and anabolism => amphibolic pathways
Cells regulate metabolism
* create or destroy transport membrane proteins
* enzyme synthesis and control
* use of environmental nutrients or synthesize from metabolites
Protein Synthesis
DNA--> Transcription of mRNA
RNA--> Translation using mRNA, tRNA, and rRNA to create a Protein
DNA ==> RNA==>Protein
Nucleotides: DNA and RNA compared
DNA
Code (genetic)
Double strand
Helix unwinds
Template for RNA, RNA polymerase
Base pairing A<==>T,
G <==>C
RNA
mRNA, tRNA, rRNA
single strand
Base pairing A<==>U, G<==>C
mRNA codons
tRNA anticodons
rRNA 2 subunits, P site (start), A site (ready next)
Bacteria do NOT have introns (non coding segments)
Many genes (~60 - 80 %) are constitutive and not regulated so that their products are made at a fixed rate. These genes are always "on" and will code for proteins (i.e. enzymes) that the cell needs for major life processes, such as those enzymes required for glycolysis. Other enzymes are regulated so that they are made only when needed. These control methods are known as induction and repression and controlthe the formation and amounts of enzymes present but do not control the activities.
Repression
inhibits gene expression by blocking RNA polymerase to initiate transcription
result: decreases the synthesis of enzymes
mediated by repressors (regulatory proeteins)
Induction
turns on transcription of a gene to synthesize protein (i.e. an enzyme)
mediated by inducers
response: enzyme induction
Control of Protein Synthesis
Bacterial Genes
* Structural Genes – genetic codes for the proteins to be used by the cell
* Operator Genes -- controls transcription (the "go or stop" signals) for the expression of the structural genes
* Promoter region – binding site for RNA polymerase on the DNA to start transcription
Entire unit above is called an operon (Operon = Operator site, Promoter site, and Structural Genes)
Two types of operons:
Inducible Operon
Starts with an active repressor binding with the operator, so the operon is "off"
When an Allosteric Inducer (such as a nutrient) blocks the repressor protein and inhibits binding to the operator, the operon is now turned "on" to allow transcription.
This process is called "inducible".
Repressible Operon:
If a repressor protein is inactive, then the operon is turned "on" and the structural genes are transcribed
When a co-repressor is available, it can then bind to the repessor protein which then binds to the operator region.
Once the repressor protein is active/activated, then the operon is turned "off" and the structural genes are not transcribed.
This process is called "repressible".
Regulatory genes control mRNA synthesis which in turn codes for the various proteins that affect the operon.
Repressor Genes – located at a distant site on the DNA and is also can be considered part of the operon system
Regulator genes that control and block the operator genes via repressor protein that can
Blocking the operator genes prevents RNA polymerase activity.
An inducer binds to repressor proteins to inactivate it and allow the RNA polymerase to bind to the promoter region to begin mRNA synthesis from a specific operon (on DNA) to start transcription.