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Lecture prepared by Mindy Miller-Kittrell, University of Tennessee, Knoxville

M I C R O B I O L O G Y WITH DISEASES BY TAXONOMY, THIRD EDITION

Chapter 5

Microbial Metabolism

Basic Chemical Reactions Underlying Metabolism

Metabolism

Collection of controlled biochemical reactions

The ultimate function of metabolism is to reproduce the organism

Basics of metabolic processes

Every cell acquires nutrients necessary for metabolism

Metabolism requires energy from light or catabolism of nutrients

Energy is stored in chemical bonds of ATP

Cells catabolize nutrients to form building blocks (precursor metabolites)

Precursor metabolites, ATP, and enzymes used in anabolic or biosynthetic reactions

Cells build macromolecules using enzymes and ATP from building blocks

Cells reproduce once they have achieved a certain size

Basic Chemical Reactions Underlying Metabolism

Catabolism and anabolism

A series of reactions in metabolism are called pathways. There are two major classes of metabolic reactions:

Catabolic pathways

Break larger molecules into smaller products

Exergonic (release energy)

Anabolic pathways

Synthesize large molecules from the smaller products of catabolism

Endergonic (require more energy than they release)

Metabolism composed of catabolic and anabolic reactions

Figure 5.1

Metabolism

Basic Chemical Reactions Underlying Metabolism

Oxidation - reduction reactions (Redox reactions)

Transfer of electrons from molecule that donates (electron donor) electron to molecule that accepts electrons (electron acceptor)

Metabolic reactions in which electrons are accepted are reduction reactions and reactions in which electrons are donated are oxidation reactions

These reactions are always coupled - always occur simultaneously

Cells use electron carriers to carry electrons (often in H atoms) from one cell location to another

There are three important electron carriers:

Nicotinamide adenine dinucleotide (NAD+)

Nicotinamide adenine dinucleotide phosphate (NADP+)

Flavine adenine dinucleotide (FAD+) → FADH2

Oxidation-reduction (Redox) reactions or redox reactions

Figure 5.2

Oxidation-reduction or redox reactions

Basic Chemical Reactions Underlying Metabolism

ATP Production and Energy Storage

Organisms release energy from nutrients

Can be concentrated and stored in high-energy phosphate bonds of ATP

Phosphorylation – organic/inorganic phosphate is added to substrate (ADP)

Cells phosphorylate ADP to ATP in three ways:

Substrate-level phosphorylation: transfer of phosphate to ADP from phosphorylated organic compound

Oxidative phosphorylation: Energy from redox (respiration) used to attach phosphate to ADP

Photophosphorylation: Light energy used to phosphorylate ADP

Anabolic pathways use some energy of ATP by breaking a phosphate bond

Basic Chemical Reactions Underlying Metabolism

The roles of enzymes in metabolism

There are six categories of enzymes based on mode of action:

Hydrolases: breakdown macromolecules by adding water in hydrolysis reaction

Isomerases: rearrange atoms within a molecule (neither catabolic nor anabolic)

Ligases or polymerases: join two molecules together (anabolic)

Lyases: split large molecule (catabolic)

Oxidoreductases: remove electrons from (oxidized) or add electrons to (reduced) substrates (catabolic and anabolic pathways)

Transferases: transfer functional groups (amino, phosphate) between molecules (anabolic)

Basic Chemical Reactions Underlying Metabolism

The roles of enzymes in metabolism (continued)

Enzymes are organic catalysts – increase the likelihood of a reaction but are not permanently changed

Many protein enzymes are complete in themselves and composed entirely of protein (chains of amino acids, folded into tertiary structure).

Some are RNA molecules called ribozymes

Others composed of protein portions called apoenzymes

Apoenzymes are inactive if not bound to non-protein cofactors (inorganic ions or coenzymes)

Binding of apoenzyme and its cofactor(s) yields holoenzyme

Makeup of a protein enzyme

Figure 5.3

Makeup of a protein enzyme

Enzyme activity

Catalyzes reactions within cells by lowering activation energy, energy needed to trigger a chemical reaction

Enzymes have functional sites (active sites) which are complementary to the shape of their substrates (molecules upon which enzymes act on)

Enzyme-substrate reaction is specific and is critical to enzyme activity

Reaction forms a temporary and intermediate compound called enzyme-substrate complex

During an enzyme-substrate reaction, chemical bonds are either broken to form new products or linked together to form a single product from two reactants

Finally, enzyme disassociates from the newly formed molecules and is ready to associate with another substrate molecule

The effect of enzymes on chemical reactions

Figure 5.4

Effect of enzymes on chemical reactions

Enzymes fitted to substrates

Figure 5.5

Enzymes fitted to substrates-overview

The process of enzymatic activity

Figure 5.6

The process of enzymatic activity

Basic Chemical Reactions Underlying Metabolism

Factors influencing the rate of enzymatic reactions

Many factors influence the rate of enzymatic reactions

Enzyme and substrate concentrations

Temperature

Presence of inhibitors

Inhibitors

Substances that block an enzyme’s active site

Do not denature enzymes

Three types of inhibitors:

Basic Chemical Reactions Underlying Metabolism

Factors influencing the rate of enzymatic reactions (continued)

Three types of inhibitors:

Competitive inhibitors: Fit into an enzyme’s active site and prevent substrate from binding. Binding results in temporary or permanent loss of enzyme activity

Noncompetitive inhibitors: do not bind to active site but prevent enzymatic activity by binding to an allosteric site. Binding at an allosteric site alters the shape of enzyme at the active site so that substrate cannot bind

Feedback (negative) inhibitors: the end-products of a series of reactions is an allosteric inhibitor of an enzyme in an earlier part of the pathway

Factors that affect enzyme activity

Figure 5.7

Effects of temperature, pH, and substrate concentration on enzyme activity

Denaturation of protein enzymes

Figure 5.8

Denaturation of protein enzymes

Competitive inhibition of enzyme activity

Figure 5.9

Competitive inhibition of enzyme activity

Allosteric control of enzyme activity

Figure 5.10

Allosteric control of enzyme activity

Feedback Inhibition

Figure 5.11

Feedback inhibition-overview

Carbohydrate Catabolism

Carbohydrate catabolism

Many organisms oxidize carbohydrates as the primary energy source for anabolic reactions

Glucose used most commonly (also used are: other sugars, amino acids and fats after first converted to glucose)

Glucose is catabolized by either:

Cellular respiration → Utilizes glycolysis, Krebs cycle, and electron transport chain; results in complete breakdown of glucose to carbon dioxide and water; large amounts of ATP produced

Fermentation → Utilizes glycolysis then converts pyruvic acid into organic fermentation products (organic waste products). Lacks Krebs cycle and electron transport chain, thus, fermentation results in the production of much less ATP

Summary of glucose catabolism

Figure 5.12

Summary of glucose catabolism

Carbohydrate Catabolism

Glycolysis (Embden-Meyerhof pathway)

Occurs in the cytoplasm of most cells

Involves splitting of a six-carbon glucose into two three-carbon sugar molecules

Direct transfer of phosphate between two substrates (PEP and ADP) occurs four times – substrate level phosphorylation.

Two ATP molecules invested by substrate level phosphorylation to lyse glucose and 4 molecules of ATP produced

Net gain of two ATP molecules, two molecules of NADH, and precursor metabolite pyruvic acid

Glycolysis is divided into three stages involving 10 total steps:

Energy-Investment Stage

Lysis Stage

Energy-Conserving Stage

Glycolysis

Figure 5.13

Glycolysis-overview

Substrate-level phosphorylation

Figure 5.14

Substrate-level phosphorylation

Carbohydrate Catabolism

Cellular respiration

Resultant pyruvic acid from glycolysis completely oxidized to produce ATP by a series of redox reactions. There are three stages of cellular respiration:

Synthesis of acetyl-CoA

Krebs cycle

Final series of redox reactions which constitute an electron transport

chain (ETC)

Synthesis of acetyl-CoA

Acetyl coenzyme A (Acetyl CoA) formed from pyruvic acid by enzymatic removal of CO2 (decarboxylation) and joining acetate to form coenzyme A

Synthesis results in:

Two molecules of acetyl-CoA

Two molecules of CO2

Two molecules of NADH

Formation of acetyl-CoA

Figure 5.15

Formation of acetyl-CoA

Carbohydrate Catabolism

Cellular Respiration

The Krebs cycle

Great amount of energy remains in bonds of acetyl-CoA

A series of eight enzymatically catalyzed reactions that transfer much of this energy to coenzymes, NAD+ and FAD+. Two carbons in acetate are oxidized and the coenzymes are reduced

Occurs in cytoplasm of prokaryotes and in matrix of mitochondria in eukaryotes. There are eight types of reactions in Krebs cycle:

Anabolism of citric acid (step 1)

Isomerization reactions (steps 2, 7 and 8)

Hydration reaction (Step 7)

Redox reactions (steps 3,4,6 and 8)

De-carboxylations (steps 3 and 4)

Substrate-level phosphorylation (step 5)

The Krebs cycle

Figure 5.16

The Krebs cycle

Carbohydrate Catabolism

Cellular Respiration

The Krebs Cycle

For every two molecules of acetyl-CoA that pass through the Krebs Cycle:

Two molecules of ATP (step 5) and four molecules of CO2 (steps 3 and 4) are produced. A molecule of guanosine triphosphate (GTP), which is similar to ATP serves as an intermediary

Redox reactions produce six molecules of NADH (steps 3, 4 and 8) and two molecules of FADH2 (step 6)

In the Krebs cycle, little energy is captured directly in high-energy phosphate bonds, but much energy is transferred via electrons to NADH and FADH2.

Carbohydrate Catabolism

Cellular Respiration

Electron transport chain (ETC)

The most significant production of ATP occurs through stepwise release of energy from a series of redox reactions between molecules known as an electron transport chain (ETC)

Consists of series of membrane-bound carrier molecules that pass electrons from one to another and ultimately to a final electron acceptor

Energy from electrons used to pump protons (H+) across the membrane, establishing a proton gradient that generates ATP via chemiosmosis

Located in the inner membranes of mitochondria (cristae) of eukaryotes and in the cytoplasmic membrane of prokaryotes

NADH and FADH2 donate electrons as hydrogen atoms (electrons and protons); whereas carrier molecules only pass the electrons down the chain

An electron transport chain

Figure 5.17

An electron transport chain

Carbohydrate Catabolism

Cellular Respiration

Electron transport chain (ETC)

Four categories of carrier molecules:

Flavoproteins: integral membrane proteins that contain a coenzyme derived from riboflavin (vitamin B2): FMN is the initial carrier and FAD is a coenzyme

Ubiquinones: lipid-soluble, non-protein carriers derived from vitamin K; in mitochondria, ubiquinone is called coenzyme Q

Metal-containing proteins: mixed group of integral proteins containing iron, sulfur and copper atoms that can alternate between the reduced and oxidized states

Cytochromes: integral proteins associated with heme, pigmented molecule found in the hemoglobin of blood

Some organisms can vary their carrier molecules under different environmental conditions: In aerobic respiration (aerobes), oxygen serves as final electron acceptor to yield water. In anaerobic respiration (anaerobes), molecules other than oxygen serve as the final electron acceptor

Possible arrangement of an electron transport chain

Figure 5.18

One possible arrangement of an electron transport chain

Carbohydrate Catabolism

Cellular Respiration

Chemiosmosis

Use of electrochemical gradients to generate ATP. Chemicals diffuse from areas of high concentration to areas of low concentration and toward an electrical charge opposite their own. The blockage of diffusion creates potential energy

Membranes maintain electrochemical gradient by keeping one or more chemicals in higher concentration on one side

Cells use energy released in redox reactions of ETC to create electrochemical gradient known as proton gradient, which has potential energy known as proton motive force.

H+ ions (protons) propelled by proton motive force, flow down electrochemical gradient through protein channels called ATP synthases (ATPase) that phosphorylate ADP to ATP

ETC is called oxidative phosphorylation because proton gradient created by oxidation of components of ETC

Total of ~34 ATP molecules formed from one molecule of glucose

Carbohydrate Catabolism

Alternative pathways to glycolysis

Yield fewer molecules of ATP than glycolysis

Reduce coenzymes and yield different metabolites needed in anabolic pathways

Two pathways:

Pentose phosphate pathway

Entner-Doudoroff pathway

Carbohydrate Catabolism

Figure 5.19

Pentose phosphate pathway

Carbohydrate Catabolism

Entner-Douoroff pathway

Carbohydrate Catabolism

Fermentation

Sometimes cells cannot completely oxidize glucose by cellular respiration

Cells require constant source of NAD+ that cannot be obtained by simply using glycolysis and the Krebs cycle

In respiration, electron transport regenerates NAD+ from NADH

Fermentation pathways provide cells with alternative source of NAD+

Partial oxidation of sugar (or other metabolites) to release energy using an organic molecule as an electron acceptor rather than ETC (NADH oxidized to NAD+ while organic molecule reduced)

Carbohydrate Catabolism

Fermentation:

In the simple fermentation reaction, NADH reduces pyruvic acid (from glycolysis) to form lactic acid. Another simple fermentation pathway involves a decarboxylation reaction and reduction results to form ethanol.

Fermentation products and organisms that produce them

Figure 5.22

Representative fermentation products and the organisms that produce them

Carbohydrate Catabolism

Other Catabolic Pathways

Other catabolic pathways

Lipid Catabolism

Protein Catabolism

Lipid and protein molecules contain abundant energy in their chemical bonds. First converted into precursor metabolites, which serve as substrates in glycolysis and the Krebs cycle.

Lipid catabolism

Fats (glycerol and fatty acids) important in ATP and metabolite production

Lipases hydrolyze bonds attaching glycerol to fatty acids

Glycerol converted to DHAP and oxidized to pyruvic acid

Fatty acids degraded by beta-oxidation and converted to acetyl-CoA

NADH and FADH2 generated during beta-oxidation are utilized in the Krebs cycle to produce ATP

Catabolism of a fat molecule

Protein Catabolism

Protein catabolism

Some microorganisms (bacteria and fungi) catabolize proteins as main source of energy and metabolites

Other cells catabolize proteins and fat only when carbon sources are not available

Proteins too large to cross cell membranes; proteases split proteins into amino acids

Amino acids further broken down by deamination and altered molecules enter the Krebs cycle

Amino groups either recycled to synthesize other amino acids or excreted as wastes

Protein catabolism

Figure 5.24

Protein catabolism in microbes

Photosynthesis

Many organisms synthesize their own organic molecules from inorganic carbon dioxide

Most of these organisms capture light energy and use it to synthesize carbohydrates from CO2 and H2O by a process called photosynthesis

Photosynthesis

Chemicals and Structures

Chlorophylls

Important to organisms that capture light energy with pigment molecules

Composed of hydrocarbon tail attached to light-absorbing active site centered on magnesium ion

Active sites structurally similar to cytochrome molecules in ETC

Structural differences cause absorption at different wavelengths

Photosynthesis

Chemicals and Structures

Photosystems

Arrangement of molecules of chlorophyll and other pigments to form light-harvesting matrices

Embedded in cellular membranes called thylakoids

In prokaryotes – invagination of cytoplasmic membrane

In eukaryotes – formed from inner membrane of chloroplasts

Arranged in stacks called grana

Stroma is space between outer membrane of grana and thylakoid membrane

Photosynthesic structures in a prokaryote

Figure 5.25

Photosynthetic structures in a prokaryote

Photosynthesis

Chemicals and Structures

Two types of photosystems

Photosystem I (PS I)

Photosystem II (PS II)

Photosystems absorb light energy and use redox reactions to store energy in the form of ATP and NADPH

Light-dependent reactions depend on light energy

Light-independent reactions synthesize glucose from carbon dioxide and water

Photosynthesis

Light-Dependent Reactions

As electrons move down the chain, their energy is used to pump protons across the membrane

Photophosphorylation uses proton motive force to generate ATP

Photophosphorylation can be cyclic or noncyclic

Reaction center of photosystem

Figure 5.26

Reaction center of a photosystem

The light-dependent reactions of photosynthesis

Figure 5.27

Photosynthesis: photophosphorylation-overview

Photosynthesis

Light-Independent Reactions

Do not require light directly

Use ATP and NADPH generated by light-dependent reactions

Key reaction is carbon fixation by Calvin-Benson cycle

Three steps

Simplified diagram of the Calvin-Benson cycle

Figure 5.28

Simplified diagram of the Calvin-Benson cycle

Other Anabolic Pathways

Other anabolic pathways

Anabolic reactions are synthesis reactions requiring energy and a source of metabolites

Energy derived from ATP from catabolic reactions

Many anabolic pathways are the reverse of catabolic pathways

Reactions that can proceed in either direction are amphibolic

Gluconeogenesis

Figure 5.29

Role of gluconeogenesis in the biosynthesis of complex carbohydrates

Biosynthesis of fat

Figure 5.30

Biosynthesis of fat

Synthesis of amino acids by amination and transamination

Figure 5.31

Synthesis of amino acids via amination and transamination

The biosynthesis of nucleotides

Figure 5.32

Biosynthesis of nucleotides

Integration and Regulation of Metabolic Function

Cells synthesize or degrade channel and transport proteins

Cells often synthesize enzymes needed to catabolize a substrate only when substrate is available

If two energy sources are available, cells catabolize the more energy efficient of the two first

Cells synthesize metabolites they need, cease synthesis if metabolite is available

Eukaryotic cells isolate enzymes of different metabolic pathways within membrane-bounded organelles

Cells use allosteric sites on enzymes to control activity of enzymes

Feedback inhibition slows/stops anabolic pathways when product is in abundance

Cells regulate amphibolic pathways by requiring different coenzymes for each pathway

Integration and Regulation of Metabolic Function

Two types of regulatory mechanisms

Control of gene expression

Cells control amount and timing of protein (enzyme) production

Control of metabolic expression

Cells control activity of proteins (enzymes) once produced

Integration of cellular metabolism

Figure 5.33

Integration of cellular metabolism

Metabolism

Oxidation-reduction or redox reactions

Makeup of a protein enzyme

Effect of enzymes on chemical reactions

Enzymes fitted to substrates-overview

The process of enzymatic activity

Effects of temperature, pH, and substrate concentration on enzyme activity

Denaturation of protein enzymes

Competitive inhibition of enzyme activity

Allosteric control of enzyme activity

Feedback inhibition-overview

Summary of glucose catabolism

Glycolysis-overview

Substrate-level phosphorylation

Formation of acetyl-CoA

The Krebs cycle

An electron transport chain

One possible arrangement of an electron transport chain

Pentose phosphate pathway

Entner-Douoroff pathway

Representative fermentation products and the organisms that produce them

Protein catabolism in microbes

Photosynthetic structures in a prokaryote

Reaction center of a photosystem

Photosynthesis: photophosphorylation-overview

Simplified diagram of the Calvin-Benson cycle

Role of gluconeogenesis in the biosynthesis of complex carbohydrates

Biosynthesis of fat

Synthesis of amino acids via amination and transamination

Biosynthesis of nucleotides

Integration of cellular metabolism