MICROBIAL BIOCHEMISTRY BIOT 309, 2012 Kim and Gadd, Chapter 4

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!!!