Chapter 5

Microbial Metabolism

Lectures prepared by Christine L. Case

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Lectures prepared by Christine L. Case

Biochemical Process
Many of the biochemical processes in bacteria also occur in eukaryotic cells However, some reactions are unique to bacteria
Use of cellulose, petroleum, inorganic carbon dioxide, iron, sulfur, hydrogen and ammonia

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Catabolic and Anabolic Reactions

Learning Objectives 5-1 Define metabolism, and describe the fundamental differences between anabolism and catabolism. 5-2 Identify the role of ATP as an intermediate between catabolism and anabolism.

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Catabolic and Anabolic Reactions

Metabolism: the sum of all the chemical reactions in an organism

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Catabolic and Anabolic Reactions

Catabolic or degradative reaction: provides energy and building blocks for anabolism Usually Hydrolytic reactions use water and breakdown chemical bonds Classified as Exergonic reactions - produce more energy than they take Such molecules that are broken down are: lipids, starches, proteins, ect. Anabolic or biosynthetic reaction: uses energy and building blocks (eg. Amino acids, fatty acids, nucleotides) to build large molecules Dehydration synthesis release water, form chemical bonds Classified as Endergonic reactions use more energy than they produce

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Catabolic and Anabolic Reactions

A metabolic pathway is a sequence of enzymatically catalyzed chemical reactions in a cell Metabolic pathways are determined by enzymes Enzymes are encoded by genes

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Check Your Understanding

Check Your Understanding Distinguish catabolism from anabolism. 5-1 How is ATP an intermediate between catabolism and anabolism? 5-2

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Enzyme
Learning Objectives 5-3 5-4 5-5 5-6 Identify the components of an enzyme. Describe the mechanism of enzymatic action. List the factors that influence enzymatic activity. Distinguish competitive and noncompetitive inhibition. 5-7 Define ribozyme.

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Collision Theory
The collision theory states that chemical reactions can occur when atoms, ions, and molecules collide
Energy transferred in collision can be used to make or break chemical bonds Whether collision results in a chemical reaction depends on: Velocity, Energy, and Chemical configuration or orientation of particles Faster the molecules move = more likely to have raction Energy must be sufficient Chemical configuration is helped manipulated by enzymes
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Collision Theory
Activation energy is the amount of energy needed to disrupt the stable electronic configurations of molecules so that the electrons can be rearranged (for chemical reaction to takes place) Reaction rate is the frequency of collisions with enough energy to bring about a reaction Reaction rate can be increased by; Heat- increases velocity of molecules, frequency of collision and number of reactant that reach activation energy Pressure,- increases frequency of collisions Concentration of reactants- increases frequency of collisions Enzymes- increase reaction rate by decreasing activation energy by orienting the molecules into a configuration that will more likely result in a chemical reaction

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Enzymes and Chemical Reactions

Catalysts- speeds up chemical reactions without being permanently altered Enzymes are biological catalysts Enzymes orients substrates into position that increases the chance of a reaction Enzymes-substrate complex increases the effectiveness of collision (decreases the activation energy) Enzymes are specific; eg. polymerase
Act on specific substrate Catalyze only one reaction
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Figure 5.2 Energy requirements of a chemical reaction.

Reaction without enzyme

Activation energy without enzyme

Reaction with enzyme Reactant Initial energy level

Activation energy with enzyme

Final energy level Products

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Enzyme Components
Biological catalysts
Specific for a chemical reaction; not used up in that reaction

Apoenzyme: protein component (inactive alone) Cofactor: non-protein component

Coenzyme: organic cofactor

Holoenzyme: apoenzyme plus cofactor (active form of the enzyme)

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Important Coenzymes
Many derive from vitamins Ivolved in atoms or electron transfer
NAD+ NADP+
-Derive from vitamin

B nicotinic acid

FAD FMN -Derive from vitamin B riboflavin

Coenzyme A

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Enzyme Specificity and Efficiency

Large globular proteins 3D shape with specific surface configuration Unique configuration (active site) enables enzyme to find and fit its specific substrate Enzymes are very efficient Can increase reaction rates up to 1010 The turnover number maximum number of substrates converted to product per second.
Is generally 1 to 10,000 molecules per second (up to 500,000)- eg. DNA polymerase has a turn over number of 15
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Enzyme Classification
Enzyme names usually end with an ase All enzymes fall into 1 of 6 groups
Oxidoreductase: oxidation-reduction reactions Transferase: transfer functional groups Hydrolase: hydrolysis Lyase: removal of atoms without hydrolysis Isomerase: rearrangement of atoms Ligase: joining of molecules; uses ATP

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Factors Influencing Enzyme Activity

Temperature- Optimal temperature facilitates reaction, high temp can denature proteins (enzymes), proteins can also be denatured by acids, bases, heavy metal ions, alcohol, and UV radiation, effects of temp. are much more drastic than pH pH- enzyme activity declines at pH below or above optimal due to 3D structure, protein denature, optimal temperature facilitates reaction Substrate concentration- if the substrate concentration is really high, there is a point where the active sites of the enzymes are all occupied (this is called saturate state); therefore, further addition of substrate will not be beneficial Inhibitors- 2 types: Competitive and noncompetitive

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Enzyme Inhibitors: Competitive Inhibition

Shape and chemical structure similar to substrate Competes with substrate for active site Inhibitors do not undergo a reaction Can be reversible or irreversible Increasing substrate concentration can overcome reversible inhibition, but not irreversible b/c the enzymes will be permanently deactivated.

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Enzyme Inhibitors: Competitive Inhibition

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Enzyme Inhibitors: Noncompetitive Inhibition

Interact with different sites of enzyme Binding causes changes to active site to change configuration and become inactive Called allosteric (other space) inhibition Reversible or irreversible Some allosteric interactions can activate enzymes Other form of noncompetitive inhibition will be the binding and depletion of cofactors (eg. Metal ions) Components needed to become a Holoenzyme are not present thus the enzyme stays an Apoenzyme
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Enzyme Inhibitors: Feedback Inhibition

Feedback or end product inhibition is a form of allosteric inhibition in which the inhibitor is the end product of a metabolic pathway. Usually inhibits the first enzyme of the pathway thus shutting of the pathway

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Ribozymes
Before 1982 all enzymes were thought to be proteins Microbiologists discovered an RNA (ribo-nucleic acid) enzyme ribozyme Ribozymes function as catalyst, have active sites that bind substrate Ribozymes cut and splices (joins) RNA RNA is not protein, it is a nucleic acid, Ribozyme is another form of RNA

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Check Your Understanding

Check Your Understanding What is a coenzyme? 5-3- organic cofactor Why is enzyme specificity important? 5-4 What happens to an enzyme below its optimal temperature? Above its optimal temperature? 5-5slow reaction, denaturation Why is feedback inhibition noncompetitive inhibition? 5-6- alosteric inhibition in wh/ the products inhibit further reactions What is a ribozyme? 5-7- an RNA that cuts and splices RNA
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Energy Production
Learning Objectives 5-8 Explain the term oxidation-reduction. 5-9 List and provide examples of three types of phosphorylation reactions that generate ATP.

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Oxidation-Reduction Reactions
ATP is the readily available high energy molecule because it has unstable phosphate bonds Oxidation: removal of electrons Reduction: gain of electrons Redox reaction: an oxidation reaction paired with a reduction reaction

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Oxidation-Reduction Reactions
In biological systems, the electrons are often associated with hydrogen atoms Hydrogen ion is made of one proton and one electron
Removal of H+ is an oxidation reaction and acceptance of H+ is a reduction reaction

Biological oxidations are often dehydrogenations

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The Generation of ATP

Energy released in redox reaction is stored as ATP ATP is a readily available high-energy molecule (has unstable phosphate bond) ATP is generated by the phosphorylation of ADP

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The Generation of ATP

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Substrate-Level Phosphorylation
Energy from the transfer of a high-energy PO4 in a phosphorylated substrate to ADP to generate ATP

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Oxidative Phosphorylation
Energy released from transfer of electrons (oxidation) of one compound to another (reduction) is used to generate ATP in the electron transport chain

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Photophosphorylation
Occurs only in photosynthetic cells Converts light energy to chemical energy of ATP and NADPH Light causes chlorophyll to give up electrons (oxidizing chlorophyll) Energy released from transfer of electrons (oxidation) of chlorophyll through a system of carrier molecules is used to generate ATP Energy is used to produce organic molecules sugar- from carbon dioxide and water
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Metabolic Pathways of Energy Production

Sequence of enzymatically catalized chemical reactions in a cell

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Check Your Understanding

Check Your Understanding Why is glucose such an important molecule for organisms? 5-8- most readily availible molecule for ATP production (first-line metabolism used in cells) Outline the three ways that ATP is generated. 5-9

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Carbohydrate Catabolism
Learning Objectives 5-11 Describe the chemical reactions of glycolysis. 5-12 Identify the functions of the pentose phosphate and Entner-Doudoroff pathways. 5-13 Explain the products of the Krebs cycle. 5-14 Describe the chemiosmotic model for ATP generation. 5-15 Compare and contrast aerobic and anaerobic respiration. 5-16 Describe the chemical reactions of, and list some products of, fermentation.
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Carbohydrate Catabolism
Most cellular ATP is from carbohydrate metabolism Glucose is most commonly used (easiest to oxidize) Two types of glucose metabolism
Respiration- complete catabolism produce a max of 38 ATP in prokaryotic cell; however, in eukaryotic cells 2 ATP are used to shuttle the pyruvic acid to the innermitochondrion membrane, thus resulting in 36 ATP Fermentation partial catabolism

The breakdown of carbohydrates to release energy

Glycolysis Krebs cycle Electron transport chain
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Glycolysis
The oxidation of glucose to pyruvic acid produces ATP and NADH Series of 10 ezymatic chemical reactions Produces 2 ATPs and 2NADHs per glucose molecule Substrate level of phosphorylation

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Alternatives to Glycolysis
Pentose phosphate pathway 1 NADPH and 1 ATP
Uses pentoses (5 carbon.glucose has 6 carbon) and NADPH Operates with glycolysis Pentose metabolism produces a lot of intermediate molecules, wh/ are used in the synthesis other essential products

Entner-Doudoroff pathway 2NADPH and 1 ATP

Produces NADPH and ATP Does not involve glycolysis Pseudomonas, Rhizobium, Agrobacterium
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Cellular Respiration
Respiration is an ATP generating process Oxidation of molecules liberates electrons for an electron transport chain The final electron acceptor is an inorganic molecule eg. Oxygen, nitrogen, ect. (In fermentation it is an organic molecule) ATP is generated by oxidative phosphorylation (34 of the total ATP) 2 types aerobic (final electron acceptor) and anerobic respiration
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Intermediate Step
Pyruvic acid (from glycolysis) is oxidized and decarboxylated into acetyl CoA

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The Krebs Cycle

Oxidation of acetyl CoA that produces NADH and FADH2

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Figure 5.13 The Krebs cycle.

1 A turn of the cycle begins

as enzymes strip off the CoA portion from acetyl CoA and combine the remaining twocarbon acetyl group with oxaloacetic acid. Adding the acetyl group produces the sixcarbon molecule citric acid.

8 Enzymes rearrange chemical bonds, producing three different molecules before regenerating oxaloacetic acid. In step 6, an oxidation produces FADH2. In step 8, a final oxidation generates NADH and converts malic acid to oxaloacetic acid, which is ready to enter another round of the Krebs cycle.
6

2 4 Oxidations generate NADH. Step 2 is a rearrangement. Steps 3 and 4 combine oxidations and decarboxylations to dispose of two carbon atoms that came from oxaloacetic acid. The carbons are released as CO2, and the oxidations generate NADH from NAD+. During the second oxidation (step 4), CoA is added into the cycle, forming the compound succinyl CoA.

5 ATP is produced by substratelevel phosphorylation. CoA is removed from succinyl CoA, leaving succinic acid.

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The Electron Transport Chain

A series of carrier molecules that are, in turn, oxidized and reduced (redox) as electrons are passed down the chain
Through these redox reactions, protons are pumped to the intermembrane space, thus creating a proton gradient eventually leading to chemiosmosis.

Energy released can be used to produce ATP by chemiosmosis in which the protons are pumped back into the matrix through an enzyme called ATP synthase wh/ converts ADP to ATP

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A Summary of Respiration
Aerobic respiration: the final electron acceptor in the electron transport chain is molecular oxygen (O2) Anaerobic respiration: the final electron acceptor in the electron transport chain is NOT O2, rather a different inorganic molecule
Yields less energy than aerobic respiration because only part of the Krebs cycle operates under anaerobic conditions

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Fermentation
Any spoilage of food by microorganisms (general use) ***not scientific Any process that produces alcoholic beverages or acidic dairy products (general use) ***not scientific Any large-scale microbial process occurring with or without air (common definition used in industry) ***not scientific

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Fermentation
Scientific definition:
Releases energy from oxidation of organic molecules Does not require oxygen Does not use the Krebs cycle or ETC Uses an organic molecule as the final electron acceptor

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Fermentation
Alcohol fermentation: produces ethanol + CO2 Lactic acid fermentation: produces lactic acid
Homolactic fermentation: produces lactic acid only Heterolactic fermentation: produces lactic acid and other compounds Differentiate the two on test

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Check Your Understanding

Check Your Understanding
What is the value of the pentose phosphate and EntnerDoudoroff pathways if they produce only one ATP molecule? 5-12 The Pentose phosphate is used in the synthesis other essential products. What are the principal products of the Krebs cycle? 5-13 reduced electron carriers How do carrier molecules function in the electron transport chain? 5-14 they allow for redox reactions and then donate their electron to the ETC Compare the energy yield (ATP) of aerobic and anaerobic respiration. 5-15 aerobic produces 38(prokaryotic) or 36(eukaryotic) ATP while anaerobic only produces significantly less than 38.
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Lipid and Protein Catabolism

Learning Objectives

5-17 Describe how lipids and proteins undergo catabolism. 5-18 Provide two examples of the use of biochemical tests to identify bacteria in the laboratory.

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Protein Catabolism

Protein

Extracellular proteases

Amino acids Organic acid

Deamination, decarboxylation, dehydrogenation, desulfurization

Krebs cycle

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Protein Catabolism

Urea

Urease

NH3 + CO2

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Biochemical Tests
Used to identify bacteria

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Clinical Focus: Human Tuberculosis Dallas, Texas

Figure A An identification scheme for selected species of slow-growing mycobacteria.

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Check Your Understanding

Check Your Understanding What are the end-products of lipid and protein catabolism? 5-17 Acetyl CoA On what biochemical basis are Pseudomonas and Escherichia differentiated? 5-18

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Figure 5.28 A nutritional classification of organisms.

Chemical

Light

Organic compounds

CO2

Organic compounds

CO2

O2

Not O2 Organic Inorganic compound compound

Yes

No

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