Basic Concepts of Cellular Metabolism and Bioenergetics Intermediary Metabolism The Chemistry of Metabolism Concepts of Bioenergetics Experimental Study of Metabolism

Metabolism Metabolism The summation of all chemical reactions in an organism. Metabolic differences are best studied by dividing all life into two categories. Autotrophs - organisms that use atmospheric CO2 as their sole source of carbon. Heterotrophs - life forms that obtain energy by ingesting complex carbon compounds .

Intermediary metabolism Metabolism relies on thousands of sequential enzymatically controlled reactions. Intermediary metabolism. Products from one reaction often become the reactant for the next - metabolites. Pathway. A series of reactions with a specific purpose. Linear - Glycolysis Branched - Amino acid biosynthesis Cyclic - Citric Acid Cycle Spiral - Fatty acid degradation

Intermediary metabolism Two paths of metabolism: Catabolism Degradation path. Complex organic molecules are degraded to simpler species. Production of energy. Anabolism Construction path. Biosynthesis of more complex organic compounds. Requires energy.

Energy, ATP and the movement of phosphate phosphoenolpyruvate P ADP 1,3-bisphosphoglycerate P creatine phosphate P Energy ATP glucose-1-phosphate P fructose-6-phosphate P ADP glucose-6-phosphate P

ATP ATP adenosine triphosphate a nucleotide composed of three basic units. adenine phosphate chain ribose CH 2 O OH N NH P -

ATP and ADP It takes energy to put on the third phosphate. Energy is released when it is removed. ADP - ATP conversions act as a major method of transferring energy. CH 2 O OH N NH P O- - ADP CH 2 O OH N NH P - ATP

Catabolic stages of metabolism Stage I Breakdown of macromolecules into their building blocks. No useful energy. Stage II Oxidation of Stage I products to acetyl CoA. Limited energy production. Stage III Oxidation of acetyl CoA to CO2 and H2O and energy.

Overview of catabolic processes Carbohydrates Fats Proteins Simple Sugars Fatty acids Amino acids Stage 1 Stage 2 Stage 3 Glycolysis ATP Pyruvate Acetyl CoA Citric acid cycle Oxidative phosphorylation ATP

Overview of catabolic metabolism ATP ADP + Pi protein amino acids polysaccharides hexoses pentoses ADP + Pi ATP ADP + Pi ATP lipids fatty acids ADP + Pi ADP + Pi ATP ADP + Pi ADP + Pi ATP ATP pyruvate ATP urea urea cycle ADP + Pi acetyl-CoA O2 ATP electron transport chain oxidative phosphorylation e- citric acid cycle CO2 ATP

Stage one Hydrolysis of food into smaller subunits. Handled by the digestive system.

Stage one Salivary glands Secrete amylase - digests starch. Stomach Secretes HCl - denatures protein and pepsin. Pancreas Secretes proteolytic enzymes and lipases - degrades proteins and fats.

Stage one Liver and gallbladder Deliver bile salts. - emulsify fat globules - easier to digest. Small intestine Further degradation. Produces amino acids, hexose sugars, fatty acids and glycerol. Moves materials into blood for transport to cells.

The chemistry of metabolism Six categories of biochemical reactions have been identified. Oxidation-reduction Group-transfer Hydrolysis Nonhydrolytic cleavage Isomerization and rearrangement Bond formation reactions using energy from ATP

Oxidation-Reduction Most common of all metabolic reactions. There are always two reactant molecules. They are readily identified by the transfer of hydrogen atoms. Enzymes involved in these reactions are oxidoreductases (dehydrogenases). AH2 + B A + BH2

Oxidation-Reduction When an atom or group is oxidized, some other species must accept the electrons. Many reactions are coupled to the coenzyme pairs. NAD+ / NADH NADP+ / NADPH FAD / FADH2

Coenzymes used in metabolism NAD+ NADH Oxidized form Reduced form of nicotinamide adenine dinucleotide. Used in REDOX reactions. It is a derivative of ADP and the vitamin nicotinamide. The reactive site is located on the nicotinamide portion of NAD+.

Coenzymes used in metabolism reactive site nicotinamide adenine ribose

NAD+

Coenzymes used in metabolism Example reactions of NAD+ General reaction Specific example - ethanol CH3CH2OH + NAD+ H CH3C=O + NADH + H+ OH O R C H + NAD + R C H + NADH + H + H alcohol dehydrogenase

Coenzymes used in metabolism FAD - flavin adenine dinucleotide. Another major electron carrier used in metabolism. It involves a two electron transfer so it picks up two hydrogen. FAD FADH2

Coenzymes used in metabolism FAD ribose adenine riboflavin Reactive site is highlighted

FAD

Coenzymes used in metabolism FAD typically reacts with different substrates than NAD+. FAD is often involved in oxidation reactions in which a -CH2 - CH2 - portion is oxidized to a double bond. O O || || CH3CH2CH2-C-S-CoA CH3CH=CHC-S-CoA FAD FADH2

Group-Transfer Reactions that involve moving a chemical functional group. Intermolecular. Transfer from one molecule to another. Intramolecular. Movement from one location to another on the same molecule. Phosphate is one of the most important groups that is transferred.

Group-Transfer Another common group to transfer is acyl group. O Coenzyme A (CoASH) will form a thioester linkage to this group, making it more active. R - C O

Acetyl - coenzyme A This molecule serves as the carrier pantothenate unit phosphorylated ADP NH 2 O O H CH3 O O N N C-CH2-CH2-N-C-C-C-CH2 O P O P O - N H HO CH3 O O - CH N H-N 2 O CH2-CH2 Sulfhydyl group S OH O CH3C P - O O O O - acetate This molecule serves as the carrier for the small molecules from digestion.

Acetyl - coenzyme A

Hydrolysis Water is used to split a single molecule into two separate molecules. Most common types of bonds to split Esters - fats Amides - proteins Glycosidic - carbohydrates

Hydrolysis Carbohydrates O OH H CH 2 HO + H2O enzyme

Hydrolysis Proteins + H | H2NCCOOH R R’ H O | || H2N - C - C - | || H2N - C - C - N - C - COOH | | H R’ enzyme + water

Hydrolysis Fats + 3 H2O C R O HO O C R H R’ R’’ OH C H C R’ O HO + C

Nonhydrolytic cleavage A class of reactions where molecules are split without the use of water. Lyases - Enzymes that accomplish this task. fructose-1,6- dihydroxyacetone glyceraldehyde bisphosphate phosphate 3-phosphate

Isomerization and rearrangement This category involves two kinds of chemical transformations: Intermolecular hydrogen atom shifts to the location of a double bond. Most prominent example is the aldose-ketose isomerization. Intramolecular rearrangements of functional groups. These are rare.

Isomerization and rearrangement C CH 2 OH H HO O CH2OH aldose cis-enediol intermediate ketose

Bond formation reactions using energy Category of biochemical bond formation reactions. All require an energy source. spontaneous DHase isocitrate oxalosuccinate -ketoglutarate

Concepts of bioenergetics Standard free energy change - Go The energy change occurring when a reaction, under standard conditions, proceeds from start to equilibrium. Equilibrium A + B C + D K’eq = [ C ] [ D ] [ A ] [ B ]

Standard free energy changes Go can be related to the equilibrium expression by: Go’ = -2.303 RT log K’eq where Go’ standard free energy change R gas constant, 8.316 J/mol T temperature, kelvin K’eq equilibrium constant

Standard free energy changes These types of measurements can be made by mixing the reactants at 1 molar, 25oC and a pH of 7 in a test tube. Unfortunately, they do not agree well with the conditions of a living cell. They do provide an estimate for comparing energy requirements among the many reactions in a cell.

Standard free energy changes Go’ = 0 System at equilibrium, no release or requirement of energy. Go’ < 0 Reaction releases energy as it approaches equilibrium. Go’ > 0 Reaction requires that energy be added to proceed in the direction indicated.

Experimental measurement of Go’ As an example, let’s determine Go’ for the isomerization of glucose-6-phosphate to fructose-6-phosphate. To start, solutions are mixed that result in an initial concentration of one molar for each species at standard conditions. At equilibrium we have: [ glucose-6-phosphate ] = 1.33 M [ fructose-6-phosphate ] = 0.67 M

Experimental measurement of Go’ K’eq = 0.67 M / 1.33 M = 0.50 Go’ = (-2.303)(8.315 J/mol)(298 K) log(0.5) = +1718 J/mol = +1.7 kJ/mol This indicates that energy is required for glucose-6-phosphate to be converted to fructose-6-phosphate -- it is not spontaneous.

Energy from ATP We can conduct a similar experiment using ATP and ADP: ATP + H2O ADP + Pi After mixing and allowing to reach equilibrium, we find that the concentration of ATP is too low to measure. We can’t directly obtain Go’ but at least we know that it must be negative.

Energy from ATP Using a coupled reaction, it is possible to measure the Go’ for ATP. Go’ kJ/mol glucose + ATP glucose-6-phosphate + ADP -16.7 glucose-6-phosphate + H2O glucose + Pi -13.8 Sum: ATP + H2O ADP + Pi -30.5 This is a relatively large amount of useful chemical energy.

Experimental study of metabolism To understand a pathway, one must know all of the details of each step. Characterization of each enzyme and coenzyme. Identification of the chemical pathway, including the substrate, intermediates, products and types of reaction. Identification of molecules and conditions that regulate the overall rate of the pathway.

Experimental study of metabolism Whole organisms. One can introduce radiolabeled materials and measure any labeled waste products. Tissue slices and cells. These have been used to uncover metabolic details. The citric acid cycle was characterized using this approach. Cell-free extracts. Cells are homogenized in a buffer to release cell components for study.