Metabolism of Microorganisms

Dr. Akepati S. Reddy School of Energy and Environment Thapar University Patiala (PUNJAB) – 147001 INDIA

Metabolic Diversity
1. Chemoheterotrophs: – Energy source: Organic compounds. – Carbon source: Organic compounds.
• Examples: Most bacteria, all protozoans, all fungi, and all animals.

2. Chemoautotrophs: – Energy source: Inorganic compounds (H2S, NH3, S, H2, Fe2+, etc.) – Carbon source: Carbon dioxide.
• Examples: Iron, sulfur, hydrogen, and nitrifying bacteria.

3. Photoheterotrophs: – Energy source: Light. – Carbon source: Organic compounds.
• Examples: Purple and green nonsulfur bacteria.

4. Photoautotrophs: – Energy source: Light. – Carbon source: Carbon dioxide.
• Examples: Plants, algae, photosynthetic bacteria

Classification based on metabolic requirements

Metabolism
Refers to the thousands of chemical reactions occurring in living organisms and characteristic of all organisms except viruses Subdivided into catabolism and anabolism • Anabolism – process of biosynthesis of complex organic molecules & structures from simpler ones – involves consumption of energy – often involve dehydration synthesis • Catabolism – degradative chemical process – involves break down of larger molecules into smaller ones – involves release energy – often involve hydrolysis processes Catabolic reactions provide the energy needed for driving anabolic reactions • energy from catabolic reactions is stored as ATP and used to drive anabolic reactions • catabolic reactions are often coupled to ATP synthesis while anabolic reactions to ATP hydrolysis • anabolic catabolic reactions are linked by ATP Metabolic pathways • Highly evolved intracellular series of metabolic reactions, each catalyzed by enzymes • may be linear, branched or cyclic (starting molecule is generated at the end of the pathway to initiate another turn of the cycle)

Metabolism = Catabolism + Anabolism

Anabolic and Catabolic Reactions are Linked by ATP in Living Organisms

Bioenergetics

Bacteria do not have mitochondria

Aerobic bacteria are Comparable to mitochondria

Enzymes
Enzymes are proteins with molecular weight ranging from a few thousands to over one million daltons • Some enzymes (conjugated enzymes or holo-enzymes) contain non-protein part (cofactor) • Cofactors can be organic molecules (coenzymes) or an inorganic molecules • Enzyme cofactors may have metal ions (iron, magnesium, copper, manganese, zinc, cobalt, magnesium, calcium, etc.) • Coenzymes are generally carriers of other molecules or ions • Some of the coenzymes are derived from vitamins (NAD and NADP from niacin, FAD from riboflavin, coenzyme A from pantothenic acid) Enzyme naming • Many enzymes are named by adding suffix ‘ase’ • substrate name (sucrase) or reaction taking place (dehydrogenase and phosphotase) or the group to which the enzyme belongs procedes the suffix • some enzymes have traditional names

Enzymes (contd..)
Enzymes can be grouped on the basis of the type reactions they catalyse (oxidoreductases, hydrolases, ligases, etc.) Enzymes can be divided into the following classes • Oxidoreductases • Transferases (functional groups are transferred) • Hydrolases (breaking bodns by adding water) • Lyases (remove groups of atmos without hydrolysis) • Isomerases (rearrange atmos within a molecule) • Ligases (joins two molecules ATP hydrolysis provides the energy needed) Catalyze chemical reactions of metabolism • Speed up the reactions upto 10 billion times • Turnover number of substrate molecules range is 1 50,000/sec. • Speeds up the reactions through decreasing the energy of activation without increasing temperature and pressure Temperature, pressure, substrate concentration, pH and several other factors influence the enzyme activity or reaction rate

Enzymes (contd..)
Enzyme specificity • Can be best explained by Lock and Key theory • Can be substrate specific (lypase for lipids, urease for urea, protease for proteins and sucrase for sucrose) • Can be specific in terms of the products into which the accepted substrates are converted • Substrates are molecules entering the enzymatically catalyzed reactions • Products are what the substrates are enzymatically converted into Enzyme activity • Enzymes have 3-D structure and one or more active sites where chemical reactions occur • 3-D structure is essential for the enzyme activity • Denaturation that usually involve breakage of hydrogen bonds and other non-covalent bonds results in loss of 3-D structure • Excessive heat and excessive pH can cause denaturation and loss of 3-D structure • Susceptible to chemical and physical changes (many chemicals destroy enzymes)

Enzymes (contd..)
Many inhibitors affect enzyme activity • Competitive inhibition – inhibitor competes for the active sites on enzyme with the substrate • Non-competitive inhibition – inhibitor binds to an allosteric site and alters the active site configuration of the enzyme • Feedback inhibition – enzyme activity is inhibited by the end product (A enzyme-1 B enzyme-2 C enzyme-3 D) – here enzyme-1 may be inhibited by product D • Feedback inhibition regulates ATP, amino acid, numcleotide and vitamin synthesis Mechanism of enzyme action • Substrate specifically binds to the active site on the surface of the enzyme and as a consequence enzyme-substrate complex is formed – can result in change of structure of the enzyme • Substrate is transformed into product by – Rearrangement of existing atoms – Breakdown of substrate molecules – Combining with other substrate molecules • Resultant products do not fit the active site and thus are released and the enzyme site becomes free for complexing with other substrate molecules

Factors that Affect Enzyme Activity: pH, Temperature, and Substrate Concentration

Lock and key theory

• Competitive inhibitor: inhibitor binds to the active site and obstruct access of substrate to the active site • Non-competitive inhibitor (by regulatory molecule): temporarily changes enzyme and alters its relative affinity for the substrate • Non-competitive inhibitor (by enzyme poison): permanently changes enzyme and renders it non-functional

Energy and ATP
ATP is stored form of microbial energy (energy currency) – the energy is stored in the chemical bond that unites phosphate group with ADP Anabolism requires input of energy and catabolism has net output of energy Microorganisms require energy for motion, reproduction, and various other activities of microorganisms energy is released (about 10,000 calories per mole) from ATP when it is broken down into ADP and phosphate Present only in living cells - can not be stored for more than a few minutes - can not be transported across cell membranes, but portable within Can be synthesized from ADP and phosphate group while using the energy present in energy compounds such as carbohydrates – reactions of catabolism supply this energy Composed of adenine molecule, ribose molecule and 3 phosphate groups Energy released during a coupled oxidation and reduction reaction if sufficient is used to form a ATP molecule – oxidation reaction is energy yielding reaction while reduction reaction is energy consuming reaction

Oxidation-Reduction (Redox)

ATP, ADP and metabolism

Catabolism
Aerobic and anaerobic respiration and fermentation
Aerobic respiration
• includes glycolysis, transition reaction, Krebs cycle and electron transport system • oxygen is electron acceptor

Anaerobic respiration
• includes glycolysis, transition reaction, Krebs cycle and electron transport system • not oxygen but nitrate or sulfate or carbon dioxide is electron acceptor

Fermentation
• Oxygen not required • Krebs cycle and electron transport system are missing • Pyruvic acid or derivatives of pyruvic acid oxidize the NADH+H to form fermentation end products

Catabolism (contd..)
Catabolism of carbohydrates
– Maltose – glucose – glycolysis – Sucrose – glucose and fructose – both into glycolysis – Lactose – glucose and galactose – galactose into a compound of glycolysis through a series of transformations – Starch – glucose - glycolysis

Catabolism of proteins • amino acids – into compounds of glycolysis and krebs cycle through deamination
– deamination of alanine produce pyruvic acid – Deamination of aspartic acid produces oxaloacetic acid – Deamination of glutamic acid produces alphaketoglutaric acid

Catabolism of fats
– Fats – glycerol and fatty acids – glycerol to dihydroxyacetone phosphate (glycolysis) – Fatty acids – beta oxidation into acetyle CoA – krebs cycle

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 2x2= 4 ATP

ATP Glycolysis 2 Transition Reaction 0 Krebs Cycle 2 E.T.S. 34 Total 38 ATP

Catabolism of various organic food molecules

Anabolism: Photosynthesis
Photoelectric effect (forcing electrons out from materials) and pigments • Particle properties of light cause it • Each substance has critical wavelength (maximum wavelength of light that creates photoelectric effect) • Pigment is any substance that absorbs light
– every pigment has a characteristic light absorption spectrum – color of the pigment is from the non-absorption but reflection of a specific wavelength light (in the visible region) by it

A process by which plants, some bacteria and some protists use light energy to produce sugar is photosynthesis • Also occurs in microorganisms having pigments (purple and green sulfur bacteria, cyanobacteria and algae) • Activities of green pigment (chlorophyll) are responsible and water and carbon dioxide are used and oxygen is released Includes two phases • use of light energy to synthesize ATP and NADPH (energy fixing or light reactions) • use these energy molecules to formulate carbohydrate molecules (carbon fixing or dark reactions)

Anabolism: Photosynthesis (contd..)
All photosynthetic organisms have chlorophyll A and also some accessary pigments other chlorophylls (chlorophyll B, C, D and E), xanthophylls and carotenoids • Chlorophyll A absorbs light from violet, blue, reddish orange and red wavelengths but not from green, yellow orange wavelengths • Accessary pigments have ability to absorb even at wavelengths left out by chlorophyll A Chlorophyll is capable of triggering chemical reaction only when it is associated with proteins embedded in membrane • bacteria have in cell membrane • Eukaryotes have in chloroplasts (membrane structures enclosing thylakoids organized into grana and stoma) • Thylakoid (flattended vesicles containing photosynthetic pigments) is the structural unit of photosynthesis Light absorption by a pigment can lead to one of the following: • Energy dissipation as heat • Fluorescence of light at longer wavelength • Triggering of photoelectric effect (or chemical reaction) Action spectrum of photosynthesis – indicates relative effectiveness of light of different wavelengths

Anabolism: Photosynthesis (contd..)
Energy fixing or light reactions
• Energy fixing reactions occur in the grana portion and carbon fixing reactions occur in the stoma portion • Chlorophyll A (and other pigments) & proteins relating to light reactions are packed into thylakoid (known as photo-system) • Photosynthetic organisms usually have two photo-systems (most prokaryotes have only one – photo-system-2) • Center of photoelectric effect in photo-system-1 is P700 (chlorophyll A), and in photo-system-2 P680 (chlorophyll A) is the center • Light energy absorbed by accessory pigments is transferred to the center of the photo-system for creation of photoelectric effect
– As a consequence of photoelectric effect electrons of the center are energized and passed through electron transport chain – Finally the electrons are absorbed by photo-system-1’s center in case of photo-system-2, and by NADP+ in photosystem-1 – Electron transport from photo-system-2 to photo-system-1 is associated with ATP synthesis (photo-phosphorylation) – Electron absorption by NADP results in the production of NADPH+H+

Anabolism: Photosynthesis (contd..)
• Loss of electrons from photosystem-2 is compensated by the electrons produced through photolysis of water
– Photolysis consumes water and produces oxygen – H+ generated from photolysis is used in the production of NADPH+H+ and O-2 generated combines and comes out as O2

• Purple sulfur bacteria uses compounds like hydrogen sulfide in place of water and emits elemental sulfur • In some eukaryotes and primitive photosynthetic bacteria cyclic photophosphorilation occurs and has only photosystem-1
– Only ATP can be synthesized by this – Occurs when additional ATP is required or when there is no NADP+, cyclic electron flow occurs

• In halobacteria (facultative aerobes) there is bacteriorhodopsin (a complex of purple pigment and membrane protein) which is also capable of ATPsynthesis

Anabolism: Photosynthesis (contd..)
Carbon fixing or dark reactions
• Recent evidence indicates that a major enzyme of dark reaction is indirectly stimulated by light (dark reaction a misnomer!) • Occurs in the stoma of chloroplasts • Carbon dioxide is captured by 5-C compound (ribulose phosphate) and forms 6-C intermediate, which splits into two 3C molecules (3-phsophoglyceric acid) • 3-phosphoglycerate through using ATP and NADPH are transformed into phosphoglyceroldehyde (PGA) • PGA is transformed into glyceroldehyde 3-phosphate (G3P) while utilizing ATP and NDAPH • Part of the G3P (2 out of 12 molecules) is used in the synthesis of glucose and other organic compounds and the rest is used to regenerate ribulose phosphate • Regeneration of ribulose phosphate involves a series of transformations and consumption of ATP. • Capturing of 6 molecules of CO2 involves consumption of 12 molecules each of of ATP and NADPH in producing G3P and 6 molecules of ATP in the regeneration of ribulose phosphate • Glucose in microorganisms can combine to form starch and can can be used in the synthesis of nucleic acid constituents or ploysaccharides for cell structures

Anabolism: Photosynthesis (contd..)
C4 plants
• Carbon fixation and Carbohydrate synthesis are separated in both space and time (CO2 fixation in the mesophyll cells and calvin cycle in the bundle sheath cells surrounding the veins) • CO2 is captured by phosphoenol pyruvate (PEP, 3-C compound) in choloroplasts of mesophyll cells to form oxaloacetic acid (OAA) • OAA is converted into malic acid and transported to bundle sheath cells and broken down back into CO2 and PEP • CO2 is used in the Calvin cycle while PEP is transported back to the mesophyll cells • Affinity of CO2 to PEP is much higher than that to ribulose phosphate • as a consequence at low CO2 levels ribulose phosphate, instead of capturing CO2, is transformed into glycoic acid by oxygen • Glycoic acid can be broken down by photorespiration

Anabolism: Photosynthesis (contd..)
Bacterial photosynthesis • Purple sulfur bacteria uses compounds like hydrogen sulfide in place of water and emits elemental sulfur • In some eukaryotes and primitive photosynthetic bacteria cyclic photophosphorilation occurs and has only photosystem-1
– Only ATP can be synthesized by this – Occurs when additional ATP is required or when there is no NADP+, cyclic electron flow occurs

• In halobacteria (facultative aerobes) there is bacteriorhodopsin (a complex of purple pigment and membrane protein) which is also capable of ATP synthesis Photoautotrophs using H2S in place of water and releasing sulfur in place of oxygen Phtoheterotrophs using organic carbon in place of carbon dioxide Chemolithoautotrophs using the bond energy of inorganic compounds in place of light energy

Amino acid biosynthesis and nitrogen fixation
• Amino acids are the building blocks of proteins. • Amino acids also serve as precursors to many important small biologically active compounds, such as nucleotides, porphyrins, lipids, and co-enzymes. • Amino acids are also used to convert nitrogen and sulfur from inorganic to organic forms. • Amino acids are involved in the regeneration of ATP to be used as energy. • During the biosynthesis and degradation of amino acids, the nitrogen and carbon molecules can be better considered separately.
– The carbon molecules of most amino acid are derived from (during biosynthesis) or entered into (during degradation) the central metabolic pathways. – The first step in amino acid metabolism usually involves either addition (during biosynthesis) or removal (during degradation) of the a amino group.

Amino acid metabolism
• Only half of the amino acids needed for protein synthesis in mammals (including humans) are synthesized from within. • Essential and non-essential amino acids. • Excess amino acids are always degraded.
– Transamination reaction to dispose of nitrogen group. – Oxidative deamination reactions.

• Removal of ammonia and urea cycle.
– Urea formation and energy requirements. – Linking of urea and TCA cycle.

• Remaining carbon skeletons enter carbohydrate metabolic pathways • Generation of energy from carbon skeletons from amino acid degradation. • Genetic disorders resulting from defects in amino acid metabolism. • Amino acids can serve as precusor for many other biologically important and active compounds.

Fatty acid synthesis
Fatty acids are synthesized in cytosol from acetyl CoA Acetyl CoA is carboxilated to malonyl CoA and then used Overall reaction is
8acetyl CoA + 14NADPH + 7ATP  Palmitate + 14NADP + 7ATP + 7Pi acetyl CoA + 7malonyl CoA + 14NADPH  Palmitate + 14NADP + 8CoA

Sequence of steps involved are 1. Binding of Acetyl CoA to cys site and Malonyl CoA to pant site of the enzyme complex 2. Condensation reaction involving decarboxilation of malonyl moity and attachment of the moity present on the cys site to the moity present on the pant site 3. Reduction of ketone group of the chain into hydroxyl group, dehydration to yield transient double bond and reduction of the resultant double bond to obtain saturated chain 4. Transfer of the growing saturated chain from pant site to cys site 5. Binding of malonyl CoA on pant site of the enzyme complex continuation from step-3 (loop of steps 2 to 5) 6. Release of the saturated fatty acid from the pant site once the length reaches 16-C

Fatty acid synthesis

Fatty acid synthesis (contd..)
Acetyl CoA required for synthesis is transported from mitochondria into the cytosol and this involves transport of citrate Elongation of fatty acid chain length beyond 16-C occurs In mitochondria and involves B-oxidation pathway running in reverse and NADPH serving as electron acceptor In the endoplasmic reticulum and uses polyunsaturated fatty acids esterified to CoA as substrate – malonyl CoA is used as donor of 2-C units Introduction of double bonds (desaturation of fatty acids) occurs later and involves consumption of NADH – desaturase enzymes are involved

Stearate + NADH +H+ +O2  Oleate + NAD+ + 2H2O
Mammalean cells are unable to introduce double bonds at certain locations – hence some polyunsaturated fatty acids like linoleic acid have become dietary essentials

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