Microbial Metabolism

Chapter 5

Metabolism

Metabolism - all of the chemical reactions within a living organism

1. Catabolism ( Catabolic )– breakdown of complex organic molecules into

simpler compounds– releases ENERGY

2. Anabolism ( Anabolic )– the building of complex organic molecules from

simpler ones– requires ENERGY

Enzymes - catalysts that speed up and direct chemical reactions

A. Enzymes are substrate specific– Lipases Lipids– Sucrases Sucrose– Ureases Urea– Proteases Proteins– DNases DNA

Enzyme Specificity can be explained by the Lock and Key Theory

E + S -----> ES ------> E + P

Naming of Enzymes - most are named by adding “ase” to the substrate

Sucrose Sucrase Lipids Lipase DNA DNase Proteins Protease removes a Hydrogen Dehydrogenase removes a phosphate phosphotase

Naming of Enzymes

Grouped based on type of reaction they catalyze

1. Oxidoreductases oxidation & reduction

2. Hydrolases hydrolysis

3. Ligases synthesis

More about Enzymes

Sometimes an enzyme needs help– Protein alone = apoenzyme– Helper molecule: cofactor

Could be inorganic like a metal ion (Fe+2) Could be organic coenzyme (like CoA, NAD)

– Apoenzyme + cofactor = holoenzyme.– Cofactors have an effect on nutrition

Bacteria have certain mineral requirements. Vitamins are cofactors that are needed in the “diet”.

Enzyme Components

2 Parts

1. Apoenzyme - protein portion 2. Coenzyme (cofactor) - non-protein

Holoenzyme - whole enzyme

Coenzymes

Many are derived from vitamins

1. Niacin– NAD (Nicotinamide adenine dinucleotide)

2. Riboflavin– FAD (Flavin adenine dinucleotide)

3. Pantothenic Acid– CoEnzyme A

Factors that Influence Enzymatic Activity

Denaturation of an Active Protein

Enzymes can be stopped

Conditions that disrupt the 3D shape– Acidic, alkaline, high salt, high temperature, etc.– These conditions thus affect growth of cell also.

Inhibitory molecules affect enzymes– Competitive inhibitors

Fit in active site but are not changed; prevent normal substrate from binding, prevent reaction.

– Non-competitive inhibitors Bind permanently to active site or other site which changes molecular

shape; prevents reaction.

– Allosteric inhibitor: temporary binding, regulates.

Competitive Inhibition

Both the substrate and the inhibitor fit into the active site, but the inhibitor isn’t altered by the enzyme. As long as the inhibitor is in the active site, the substrate cannot enter the active site and react. The more inhibitor molecules that are present, the more often one of them occupies the active site

.

ghs.gresham.k12.or.us/.../ competitiveinhib.htm

Allosteric sites

In allosteric site, inhibitor is not reacted, but causes a shape change in the protein. The substrate no longer fits in the active site, so it is not chemically changed either.

ghs.gresham.k12.or.us/.../ noncompetitive.htm

Competitive Inhibitors -compete for the active site

1. Penicillin – competes for the active site on the enzyme involved

in the synthesis of the pentaglycine crossbridge

2. Sulfanilamide (Sulfa Drugs)– competes for the active site on the enzyme that

converts PABA into Folic Acid Folic Acid - required for the synthesis of DNA and RNA

Selective Toxicity

Non-competitive Inhibitors - attach to an allosteric site

Feedback Inhibition- stops the cell from wasting chemical resources by making more of a substance than it needs.

Energy Production

1. Oxidation– refers to the loss of Hydrogens and or electrons

2. Reduction– the gain of Hydrogens and or electrons

NAD Cycle

Carbohydrate Catabolism

Microorganisms oxidize carbohydrates as their primary source of energy

Glucose - most common energy source Energy obtained from Glucose by:

– Respiration– Fermentation

Aerobic Cellular Respiration

Electrons released by oxidation are passed down an Electron Transport System with oxygen being the Final Electron Acceptor

General Equation:

Glucose + oxygen----> Carbon dioxide + water ATP

Chemical Equation

C6H12O6 + 6 O2 -------> 6 CO2 + 6 H2O 38 ADP + 38 P 38 ATP

Aerobic Cellular Respiration

4 subpathways

1. Glycolysis 2. Transition Reaction 3. Kreb’s Cycle 4. Electron Transport System

1. Glycolysis (splitting of sugar)

Oxidation of Glucose into 2 molecules of Pyruvic acid

Embden-Meyerhof Pathway

End Products of Glycolysis:– 2 Pyruvic acid– 2 NADH2

– 2 ATP

2. Transition Reaction

Connects Glycolysis to Krebs Cycle

End Products:– 2 Acetyl CoEnzyme A– 2 CO2

– 2 NADH2

3. Krebs Cycle (Citric Acid Cycle)

Series of chemical reactions that begin and end with citric acid

Products:– 2 ATP– 6 NADH2

– 2 FADH2

– 4 CO2

4. Electron Transport System

Occurs within the cell membrane of Bacteria

Chemiosomotic Model of Mitchell– 34 ATP

How 34 ATP from E.T.S. ?3 ATP for each NADH2

2 ATP for each FADH2

NADH2

Glycolysis 2 T. R. 2 Krebs Cycle 6

Total 10

10 x 3 = 30 ATP

FADH2

Glycolysis 0 T.R. 0 Krebs Cycle 2

Total 2

2 x 2 = 4 ATP

Total ATP production for the complete oxidation of 1 molecule of glucose in Aerobic Respiration

ATP Glycolysis 2 Transition Reaction 0 Krebs Cycle 2 E.T.S. 34

Total 38 ATP

Overview of aerobic metabolism

Energy is in the C-H bonds of glucose. Oxidation of glucose (stripping of H from C atoms) produces

CO2 and reduced NAD (NADH)– Energy now in the form of NADH (“poker chips”)

Electrons (H atoms) given up by NADH at the membrane, energy released slowly during e- transport and used to establish a proton (H+) gradient across the membrane

– Energy now in the form of a proton gradient which can do work.– Electrons combine with oxygen to produce water, take e- away.

Proton gradient used to make ATP– Energy now in the form of ATP. Task is completed!

Definitions

Substrate level phosphorylation– Chemical reaction coupled to ATP synthesis

Oxidative (respiratory) phosphorylation– Pumping of protons powered by electron transport

with oxygen as terminal electron acceptor yields ATP

Photophosphorylation– Pumping of protons powered by absorption of light.

Central Metabolism:Funneling all nutrients into central pathways

•Many other molecules besides glucose can serve as a source of carbon.

Central Metabolism:A source of building blocks for biosynthesis

BUT, these molecules can’t be broken down to CO2

for energy AND used for biosynthesis

Other ways to make ATP

Photosynthesis: light driven ATP synthesis. Anaerobic respiration: organic compounds

oxidized, electrons passed down e- transport chain to some molecule other than oxygen (e.g. NO3, SO4).

Inorganic molecules can be oxidized with ATP synthesis by e- transport and chemiosmosis.

Fermentation: common anaerobic pathway used by many medically important bacteria.

Anaerobic Respiration

Electrons released by oxidation are passed down an E.T.S., but oxygen is not the final electron acceptor

Nitrate (NO3-) ----> Nitrite (NO2-)

Sulfate (SO24-) ----> Hydrogen Sulfide (H2S)

Carbonate (CO24-) -----> Methane (CH4)

Fermentation

Anaerobic process that does not use the E.T.S. Usually involves the incomplete oxidation of a carbohydrate which then becomes the final electron acceptor.

Glycolysis - plus an additional step

Fermentation may result in numerous end products

1. Type of organism

2. Original substrate

3. Enzymes that are present and active

1. Lactic Acid Fermentation Only 2 ATP End Product - Lactic Acid Food Spoilage Food Production

– Yogurt - Milk– Pickles - Cucumbers– Sauerkraut - Cabbage

2 Genera:– Streptococcus– Lactobacillus

2. Alcohol Fermentation

Only 2 ATP End products:

– alcohol– CO2

Alcoholic Beverages Bread dough to rise

Saccharomyces cerevisiae (Yeast)

3. Mixed - Acid Fermentation

Only 2 ATP End products - “FALSE”

Escherichia coli and other enterics

Propionic Acid Fermentation

Only 2 ATP End Products:

– Propionic acid– CO2

Propionibacterium sp.

Fermentation

Figure 5.18b

Lipid Catabolism

Protein Catabolism

Biochemical tests

Figure 10.8

Used to identify bacteria.

Photosynthesis - conversion of light energy from the sun into chemical energy

Chemical energy is used to reduce CO2 to sugar (CH2O)

Carbon Fixation - recycling of carbon in the environment (Life as we known is dependant on this)

Photosynthesis– Green Plants – Algae– Cyanobacteria

Chemical Equation

6 CO2 + 6 H2O + sunlight -----> C6H12O6 + 6 O2

2 Parts:– 1. Light Reaction– 2. Dark Reaction

Light Reaction

Non-Cyclic Photophosphorylation– O2

– ATP– NADPH2

Light Reaction (simplified)

2. Dark Reaction

Macronutrients Carbon (CO2 or organic compounds) Hydrogen (H2O or organic compounds) Oxygen (H2O or organic compounds) Nitrogen (NH3, NO3

-, organic N-compounds) Phosphorus (PO4

3-) Sulfur (H2S, SO4

2-, organic compounds) Potassium (K+) Magnesium (Mg2+, salts) Sodium (Na+) Calcium (Ca2+, salts) Iron (Fe3+, Fe2+, or salts)

Iron as a nutrient

Needed for aerobic metabolism (cytochromes, iron-sulfur proteins)

Insoluble under aerobic conditions– Fe(OH)3, FeOOH

– Solubilized by siderophores

Micronutrients and growth factors

Micronutrients: Metals and metalloids– Generally not necessary to add to medium– Deficiencies can arise when medium constituents

are very pure

Growth factors: organic requirements– Vitamins, amino acids, purines, pyrimidines,

acetate

Culture media

Defined: all chemicals are ostensibly known Complex (undefined): contains substances

with unknown chemistries, such as peptones, yeast extract, lake water, soil extract, etc.