MICROBIAL BIOCHEMISTRYBIOT 309, 2012
Kim and Gadd, Chapter 4
OVERVIEW OF BACTERIAL METABOLISM
BACTERIAL METABOLISM• Metabolism = all biochemical reactions taking place in
organismConversion (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
COMPARISONAnabolism Catabolism
Buildup of bigger molecules
Breakdown of larger molecules
Products are large molecules
Products are small molecules
Protein, peptidoglycan, DNA, RNA
Glycolysis, citric acid cycle
Mediated by enzymes Mediated by enzymesE required (endergonic)
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/reaction
– 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 1Group Translocation – phosphotransferase system
ANIMATION:
http://highered.mcgraw-hill.com/sites/9834092339/student_view0/chapter5/active_transport_by_group_translocation.html
ANIMATION:http://www.microbelibrary.org/images/kaiser/
grouptranslocat.html
ANIMATIONS FOR GROUP TRANSLOCATION
GLYCOLYSIS, step 2
Rearrangement/change reaction, requires Mg2+
GLYCOLYSIS, step 3phosphorylation
NOTICE use of ATP QUESTION: What type of reaction is this?
GLYCOLYSIS, step 4cleavage
Yield 2 G3P
END OF PREPARATORY
PHASE Aldol cleavage
Aldose to ketose isomerization
GLYCOLYSIS, step 5coupled oxidation + phosphorylation
☐ ~ = high energy bond
Question: What is oxidized? What is reduced?
QUESTION: Where is energy of
NADH used? Where does
it go?
GLYCOLYSIS, step 6dephosphorylation
Example of substrate level phosphorylation
QUESTION: From what carbon atom is the Pi removed?Why is this Pi removed?
GLYCOLYSIS, step 7phosphate group shift
GLYCOLYSIS, step 8dehydration
~ = high energy bond
QUESTION: why does this reaction create ~Pi?
GLYCOLYSIS, step 9dephosphorylation
~ = 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 REACTIONConnects 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 COENZYMECoenzyme 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
2 carbon
Co A
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 enzymes1 Glucose 2 pyruvate + 1 ATP + 1 NADH
+ 1 NADPHBacteria: 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!!!