MICROBIAL BIOCHEMISTRY
BIOT 309, 2012
Kim and Gadd, Chapter 4
OVERVIEW OF
BACTERIAL METABOLISM
BACTERIAL METABOLISM
• Metabolism = all biochemical reactions taking place
in organism
Conversion (change, rearrangement) reactions
• One molecule becomes another
• Structure changes
• Not use or generate energy
Anabolism uses endergonic reactions
• Uses energy
• Forms bonds
• builds larger molecules, ie, proteins,
carbohydrates
BACTERIAL METABOLISM
Catabolism is exergonic
• Releases/produces energy, i.e.,
makes ATP
• Breaks bonds
• Hydrolyzes larger molecules into
simpler molecules
COMPARISON
Anabolism
Catabolism
Buildup of bigger
molecules
Products are large
molecules
Protein, peptidoglycan,
DNA, RNA
Mediated by enzymes
E required
(endergonic)
Breakdown of larger
molecules
Products are small
molecules
Glycolysis, citric acid
cycle
Mediated by enzymes
E released (exergonic)
BACTERIAL METABOLISM
• Growth depends on metabolism
• All 3 types of reactions happening at the same time
but anabolism or catabolism dominate at different
phases of growth
• Carried out by wide variety of enzymes and co-factors
• Involves single enzymes and enzyme complexes
• Provides precursor metabolites to anabolic pathways
• Occurs in three locations:
– Cytosol
– On or in cell membrane
– In periplasmic space
ENZYMES
• Characteristics
– Reusable
– Very specific – one reaction/enzyme
– Minute amounts needed
– Work fast (100-1000 reactions/minute)
– Catalysts
– Large proteins
Cont’d
ENZYMES
• Characteristics
–1
enzyme/reacti
on
– Substrate
specificity
– “Lock and Key”
“LOCK AND KEY”
“LOCK AND KEY”
with coenzyme*
* See descriptions in
White
*
*
ENZYMES
– Active site aligns substrate(s) so reaction is highly
favorable
Free energy = G
ENZYMES
• Primarily proteins
• Some have co-factors; co-factor use
based on needs of enzyme
– Inorganic ions: Mg++, Fe++, Zn++
– Organic ions: important in redox reactions
• NAD+ : EMP glycolysis, ED – Entner Duodorff pathway
• NADP+: HMP – hexose monophosphate pathway,
glycolysis
– Both inorganic and organic used in reactions in
TCA cycle and ETC (electron transport)
NAD+ (oxidized)
NADH (reduced)
As NAD+ is reduced, one electron is added at the Nitrogen atom
(removing the + charge), (= becomes - ) and one (electron +
proton = H atom) (= becomes -) is added at the upper position of
the nicotinamide ring.
ENERGY STORAGE DURING
CATABOLISM
• Must be available as energy for anabolism
• Forms of storage:
– Held in high energy bonds, e.g., ATP
– Reducing equivalents, such as NADH, NADPH & FADH2
– Proton gradient (formed by electron transport system)
• Forms used depend on pathway/enzymes used
by bacteria
• ATP and NADH are most common
COUPLED REACTIONS OCCUR: BE ABLE TO
IDENTIFY THEM
Also Important in Glycolysis and
Kreb’s Cycle!!!
• Substrate-level phosphorylation
HOMEWORK: FIND EXAMPLES FROM SLIDES AND TEXTBOOK
ADDITIONAL REDOX MOLECULES
• Used in Electron Transport – Ch 5
– Ubiquinone
– Iron-sulfur
• Will review then
ENZYMES OCCUR AS:
• Single enzymes
• Part of complexes with other proteins and
cofactors *
– Electron transport chain
– Flagella
– ATP synthase
• Part of pathways
– Glycolysis
– Citric Acid Cycle
– Etc.
Slowest reaction is
a rate limiting step
BACTERIA: FOCUS ON CATABOLISM
Catabolism is exergonic
• Releases/produces energy, i.e., makes
ATP
• Hydrolyzes larger molecules into
simpler molecules
• Breaks bonds
3 phases of catabolism: glycolysis,
Kreb’s Cycle, Electron Transport
Chain (ETC)
BIG
PICTURE
Integrating
3 Phases of
Catabolism
• Reaction Products have abbreviated names
• Watch for their use and know to what they
refer
GLYCOLYSIS
• Occurs mainly in cytoplasm
– 1st step in some bacteria occurs in membrane
• Involves how many enzymes? TEN but two
ways to make glucose-6-phosphate (See STEP
1 slide.)
• Splits glucose
• NOTE: Does not require O2, i.e., this stage is
anaerobic
OVERALL GLYCOLYSIS* REACTION
glucose (6C) + 2 NAD+ + 2 ADP +2 Pi
2 pyruvate (3C) + 2 NADH + 2 H+ + 2 H2O + 2 net
ATP
Is NAD+ the oxidized or reduced form?
*also called Embden-Myerhof-Parnas Pathway
GLYCOLYSIS AND ALTERNATIVES
• Bacteria use 3 different pathways to convert
glucose to PGA (3-phosphoglycerate) (see diagram)
– Glycolysis/Embden-Myerhof-Parnas (shown in next
slide)
– Pentose phosphate shunt/hexose monophosphate
shunt
– Entner-Duodorff
– Energy yields are different
• Same pathway (transition or bridging reaction)
takes PGA (3-phosphoglycerate) to pyruvate
GLYCOLYSIS
3 different
glycolytic
pathways
operate: EMP,
EDP, HMP
 THIS IS EMP
From PGA on
same steps
GLYCOLYSIS
PHASES
Preparatory
Phase
Payoff Phase
HIGH ENERGY COMPOUNDS
• ATP
• Pyruvate
• HOMEWORK: WHAT OTHER HIGH ENERGY
COMPOUNDS ARE PART OF EMP PATHWAY?
GLYCOLYSIS, step 1
•
Rapid reaction to keep glucose inside cell
– Location 1 = cytoplasm; one enzyme = hexokinase, requires Mg2+
Glucose
glucose-6-phosphate (G6P)
– Location 2: membrane (Some bacteria)
PEP
pyruvate provides ~P to phosphorylate and transport glucose across the membrane
• More proteins and enzymes are involved
• Other sugars use similar mechanism
phosoenolpyruvate: sugar phosphotransferase system (PTS)
PEP + SUGAR
PYRUVATE + sugar-phosphate
In E.coli the PTS consists of two enzyme and a low molecular weight heat-stable protein (HPr)
WHAT DOES PEP stand for?
GLYCOLYSIS, step 1
Group Translocation – phosphotransferase
system
ANIMATIONS FOR
GROUP TRANSLOCATION
ANIMATION:
http://highered.mcgrawhill.com/sites/9834092339/student_view0/chapter5/active_
transport_by_group_translocation.html
ANIMATION:
http://www.microbelibrary.org/images/kaiser/groupt
ranslocat.html
GLYCOLYSIS, step 2
Rearrangement/change reaction, requires Mg2+
GLYCOLYSIS, step 3
phosphorylation
NOTICE use of ATP
QUESTION: What type of reaction is this?
GLYCOLYSIS, step 4
cleavage
Aldose to ketose
isomerization
Yield 2 G3P
END OF
PREPARATORY
PHASE
Aldol cleavage
GLYCOLYSIS, step 5
coupled oxidation + phosphorylation
QUESTION:
Where is
energy of
NADH used?
Where does
it go?
☐
~ = high
energy
bond
Question: What is oxidized?
What is reduced?
GLYCOLYSIS, step 6
dephosphorylation
Example of substrate level phosphorylation
QUESTION: From what carbon atom is the Pi removed?
Why is this Pi removed?
GLYCOLYSIS, step 7
phosphate group shift
GLYCOLYSIS, step 8
dehydration
~ = high energy bond
QUESTION: why does this reaction create ~Pi?
GLYCOLYSIS, step 9
dephosphorylation
~ = high energy bond
OVERALL GLYCOLYSIS* REACTION
glucose (6C) + 2 NAD+ + 2 ADP +2 Pi
2 pyruvate (3C) + 2 NADH + 2 H+
+ 2 net ATP + 2H2O
WHICH is oxidized and which is reduced?
NAD+ is __________ ; NADH is ___________
*also called Embden-Myerhof-Parnas Pathway
THE OTHER GLYCOLYTIC PATHWAYS
TRANSITION OR BRIDGING REACTION
Connects glycolysis to citric acid/Kreb’s Cycle
OVERALL REACTION
2 pyruvate + 2 NAD+ + 2 CoA-SH (coenzyme A)
2 acetyl-CoA + 2 NADH + 2 H+ + 2 CO2
CONNECTION TO OTHER BIOLOGY: Where else is
CO2 made?
NAD+ (oxidized)
NADH (reduced)
As NAD+ is reduced, one electron is added at the Nitrogen atom (removing
the + charge), (= becomes - ) and one (electron + proton = H atom) (=
becomes -) is added at the upper position of the nicotinamide ring.
ANOTHER COENZYME
Coenzyme A
Energy generation
• Molecule made from several component parts – complex
• Highly polar
• Key in glycolysis to Kreb’s cycle transition reaction
• Key component in fatty acid reactions
• Synthesis very similar pro- and eukaryotes
TRANSITION REACTION
3 carbon
Co A
2 carbon
NEXT: MORE ON TCA CYCLE
Operate
under
different
growth
conditions
Note
energy
yields
NOTE: MORE SLIDES WILL BE ADDED
ON ED AND PPS PATHWAYS
ENTNER-DUDOROFF PATHWAY
• The Entner-Doudoroff pathway yields one ATP
and two NADPH molecules from one glucose
molecule.
• Uses totally different enzymes
1 Glucose
2 pyruvate + 1 ATP + 1 NADH
+ 1 NADPH
Bacteria: Pseudomonas, Rhizobium,
Azotobacter, Agrobacterium, Enterococcus
faecalis
PENTOSE PHOSPHATE SHUNT
PATHWAY
• Precursors to the ribose and deoxyribose in nucleic acids
• Provides erythrose phosphate which is a precursor for
synthesis of aromatic amino acids
• reducing power = NADPH
Overall reaction
6 Glucose 6 – P + 12 NADP+ + 6 H2O + 5 Glucose 6 – P
+
6 CO2 + 12 NADPH + 12 H+
• Used exclusively by Thiobacillus novellus and Brucella
abortus
Pentose Phosphate
Shunt Pathway
What do abbreviations
stand for?
MAKE a list!!!