4 bio265 metabolism instructor dr di bonaventura

An overview of Microbial metabolism

What is Metabolism?

Catabolism (breakdown) Provides energy and building blocks for anabolism and other cellular functions

Anabolism (biosynthesis) Uses energy and building blocks to build larger molecules, macromolecules, and cell structures

Metabolism is composed of Catabolism and Anabolism

The sum of controlled chemical reactions that occur within a cell (microbial cell)

The role of ATP as an intermediate between catabolism and anabolism

Metabolic pathway - sequence of chemical reactions that occur in a cell

A B C D E

A is the initial molecule/compound (substrate) E is the final molecule/compound (product) B, C, and D are intermediates

Each step in the pathway is mediated or facilitated by

a specific catalyst called enzyme (protein)

Catabolic and anabolic reactions are organized into series of reactions called pathways

Cells use redox reactions to extract energy from nutrient molecules such as glucose

Redox reaction (oxidation-reduction reaction)

Redox reactions involve the transfer of electrons from an electron donor to an electron acceptor

The energy of the reduced coenzyme NADH (or FADH2) is used to

make ATP in later reactions

Cells use redox reactions to extract energy from nutrient molecules such as glucose

Most biological oxidations involve the loss of hydrogen atoms (one electron plus one proton-H+) - dehydrogenation reactions

Enzymes are the catalysts of biological reactions

Specific for a given substrate/chemical reaction

The shape of the molecule provides a distinctive site called the active site or catalytic site of the enzyme

The substrate specifically fits into the enzyme’s active site

Examples of enzymes and their names

Enzymes can use cofactors and coenzymes

Cofactors include magnesium, manganese, iron, copper, zinc, calcium, cobalt

Coenzymes (NAD+, NADP+, and FAD derived from vitamins) act as electron carriers

Enzymes lower the activation energy needed to “trigger a chemical reaction”

The substrate interacts with the active site of the enzyme to form an enzyme-substrate complex

The substrate is transformed into product(s) The product(s) is/are released The enzyme is recovered unchanged

Factors that influence the activity of enzymes include Temperature pH Enzyme and substrate concentration Presence/absence of inhibitors

Temperature and pH denature proteins, therefore enzymes

Inactive

Cells control synthesis of enzymes (amount/time of synthesis) and their activity

Enzyme inhibitors

Competitive inhibition

Sulfa drugs are an example of competitive inhibitors

Sulfanilamide/PABA

Inhibition can be reversible or irreversible

Noncompetitive inhibition

Poisons such as fluoride are an example of noncompetitive inhibitors

Enzyme inhibitors

Inhibition can be reversible or irreversible

Feedback Inhibition

It is reversible

When the final product accumulates

It begins to bind to and inactivates the enzyme that catalyzes the first reaction of the pathway

The pathway is turned off

Cells regulate metabolic

pathways

Carbohydrate Catabolism

Most microorganisms oxidize carbohydrates as the primary source of cellular energy

Two general processes are used Cellular respiration Fermentation

Both cellular respiration and fermentation can share a common pathway called glycolysis or Embden-Meyerhof pathway

Aerobic cellular

respiration Glucose (C6H12O6) is oxidized to CO2 in presence of O2

~ 38 molecules of ATP are formed

Fermentation

Releases energy from oxidation of organic molecules, i.e.; Sugars Organic acids Amino acids Purines and pyrimidines

Does not use the Krebs cycle or the electron transport chain

Uses pyruvate as the final electron acceptor from NADH, regenerating NAD+ for glycolysis

Does not require oxygen

End-Products of Fermentation

Chemical analyses of the end-products help identify microbes, including pathogens in clinical specimens

Lactic acid and alcohol Fermentation

Lactic acid bacteria are Streptococcus and Lactobacillus

Alcohol fermentation carried out by Saccharomyces cerevisiae (yeast)

How do we use this knowledge to distinguish between bacteria in the lab?

Fermentation test tubes containing mannitol

Negative control

Samples

S. epidermidis (-) S. aureus (+) E. coli (+) plus gas Bubbles in the Durham tube indicate gas formation

Microorganisms catabolize complex sugars and lipids

Microbes produce extracellular enzymes Amylases to break down

starch

Lipases to break down fats into glycerol and fatty acids

Microorganisms catabolize proteins

Microbes produce proteases

Break down proteins into their component amino acids, transported across the plasma membrane

Amino acids are chemically modified by deamination reaction

How do we use this knowledge to distinguish between bacteria in the lab?

Biochemical tests are designed to detect the presence of enzymes The test tubes contain

glucose pH indicator amino acid

Negative control Sample

pH indicator turns to yellow if acid is produced from glucose pH indicator turns to purple if alkaline products are produced from

amino acid (decarboxylation reactions)

How do we use this knowledge to distinguish between bacteria in the lab?

Production of H2S

Salmonella (+) can be readily distinguishable from E. coli (-) by the production of hydrogen sulfide

H2S is detected when bacteria remove sulfur from certain amino acids

Other tests take advantage of different components of the electron transport chain (oxidase test – cytochrome oxidase)

Anabolic pathways and their link to catabolic pathways

ATP made during catabolic pathways is used for cellular functions such as Active transport across plasma membranes Flagellar motion

Most of the ATP is used to synthesize new cellular components Amino acids are needed to make proteins/enzymes Carbohydrates needed for polysaccharides/peptidoglycan Lipids are important components of cell membranes Purines and pyrimidines are the building blocks of DNA and

RNA

Anabolic pathways and their link to catabolic pathways

The biosynthesis of simple lipids

Glycolysis and Krebs cycle provide intermediates (precursor metabolites) for anabolic pathways

Metabolic pathways that function in both anabolism and catabolism are called amphibolic pathways

Amphibolic pathways