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Matabolic Stoichiometry and Energetics in Microorganisms Dr. A.K.M. Shafiqul Islam Metabolism A living cell is a complex chemical reactor in which more than 1000 independent enzyme-catalyzed reaction occurs – The total of all chemical reaction activities which occur in the cell is called metabolism. The metabolic reaction tend to be organized into sequences called metabolic pathways which connect one reaction with another Important Coenzymes NAD+ NADP+ FAD Coenzyme A A cell produces order from its disorderly surrounding things Energy from the environment is used to drive the metabolic process In bioprocess engineering, the energy exchanges helps explain the major distinction between cell function in the presence and absence of oxygen Types of Metabolism Three types of metabolism – Aerobic Use free oxygen – Anaerobic Do not use free oxygen – facultative anaerobes A third class of cells can grow in either environment and known as facultative anaerobes. Yeast is a familiar example of this metabolic variety Two different kinds of energy are tapped by inhabitants of microbial world – Light Organism which relay on light are called phototrophs – Chemical While chemotrophs extract energy by breaking down certain nutrients. Further subdivision of chemotrophs is possible – Lithotrophs Oxidize organic materials – Organotrophs Employ organic nutrients for energy production The energy obtained from the environment is stored and shuttled in the high-energy intermediates such as ATP. Cell use this energy to perform three types of work: – chemical synthesis of large or complex molecules – transportation of ionic or neutral substances into or out of the cell or its internal organcells – mechanical work required for cell division and motion All these processes are (by themselves) nonspontaneous and result in an increase of free energy of the cell. They occur when simultaneously couple to another process which has a negative free-energy change of greater magnitude. In order to grow and reproduce, cells must ingest the raw materials necessary to manufacture membrane, protein, walls, chromosomes and other components Four major requirements are evident: – carbon, nitrogen, sulfur and phosphorus Reactions within the cell have been subdivided into three classes: – degradation of nutrients – biosynthesis of small molecules – biosynthesis of large macromolecules Each reactions are catalyzed by an enzyme. The enzyme serve the essential function of determining which reaction occur and their relative rates Thermodynamic Principles To get an idea of whether a certain reaction in the cell will run forward or backword, we will use a number of approximation since full analysis of metabolic network is not practical. First we consider the free-energy change of a chemical reaction A B C D We can write c d 0' G G RT ln a b (1) (2) In a closed system, the reaction will proceed left to right if and only if G‘ is negative. Accordingly, G‘ is zero at equilibrium give the following realtionship (3) G 0' RT ln Keq where K eq ceq d eq aeq beq (4) If water or H+ are involve in the reaction, their concentrations do not enter into the calculation of the right hand side (4). The value already includes the water and H+ concentration (for pH 7) Consider the reaction between two isomers in the Embden-Mayerhof pathway for glucose breakdown CHO CH2 OH CHOH CH2 O C P Glyceraldehyde 3-phosphate G0’= -1830 cal/mol O CH2 O P Dihydroxyacetone-P Where P denotes phosphate. Because of the negative free energy change, equilibrium favors the dihydroxyacetone by a 22:1 ratio. Many biological reaction and energy conversion process involve oxidation-reduction reaction such as Aox Bred A red Box This type of reaction is described using the standard potential change 0 ΔE E 0 Aox A red E 0 BBox Bred where E 0A ox Ared is the standard half-cell potential for the half reaction A ox 2e A red As a reference point for half-cell potential value, the hydrogen half-cell (at pH=0) is assigned a value of zero: 2H 2e H 2 E 0 0.000V (pH 0) The free energy change and corresponding potential changes are related by G nFE Where n is the number of electrons transferred and F is equal to 23.062 kcal/V mol Metabolic Reaction Coupling: ATP and NAD ATP Energy is released as food is oxidized Used to form ATP from ADP and Pi ADP + Pi + Energy ATP In cells, energy is provided by the hydrolysis of ATP ATP ADP + Pi + Energy The enzymatic hydrolysis of ATP to yield ADP and inorganic phosphate has a large negative freeenergy change ATP + H2O ADP + Pi G0’ = -7.3 kcal/mol Where Pi indicate inorganic phosphate A substantial amount of free-energy may be released by the hydrolysis By reversing the reaction and adding the phosphate to ADP, free energy can be stored for late use Embden-Meyerhof-Parnas pathway serves to illustrate the concept of a common chemical intermediate 1. Oxidation of aldehyde to carboxylic acid RCHO H 2O 2H RCOO H G10 7kcal/mol 2. Same reactions, coupled to ATP generation (glucose oxidation) RCHO HPO 4 ADP 3 2H RCOO ATP 4 G 02 0kcal/mol 2- 3. Reaction 2 and 1 yield ADP 3 HPO 4 H ATP 4 H 2O 2- 0 3 G 7kcal/mol Example OH H O = O P O C C C H + HPO4 O H OH O- H H O O2H + H2O + -O P O C C C O P OO H OH O OH H O OO P O C C C O P OO H OH O + ADP3- RCOO- + ATP4- Thus glucose metabolism is the process at which cell generates the ATP needed for endergonic process This generation is accomplished by the conversion of a partially metabolized nutrient into a high-energy phosphorylated intermediate, which then donates a phosphate to ADP via an enzyme-catalyzed reaction The phosphorylation of various compounds serves several functions It provides a useful means of storing considerable fractions of free energy of fuel oxidation. Free energies of hydrolysis of several called phosphate donors are greater than G0’ for ATP hydrolysis. Example, phosphoenolpyruvate G0’ = -14.8 kcal mol-1. 1,3-diphosphoglycerate G0’ = -11.8 kcal mol-1 Hydrolysis of this compounds can be used to drive ADP phosphorylation Similarly, ATP hydrolysis serves to phosphorylate “low energy” phosphate compounds. Example, glucose-6-phosphate G0’ = -3.3 kcal mol-1 glycerol-1-phosphate G0’ = -2.2 kcal mol-1 Highly ionized organic substances are virtually unable to permeate the cell’s plasma membranes. The charged phosphorylated compounds which serves as metabolic intermediates may therefore be contained within the cell. Thus maximum amounts of energy and chemical raw materials can be extracted from a nutrient. Oxidation reduction: Coupling via NAD Oxidation-reduction reactions are conducted biologically and the connection between these mechanisms and ATP metabolism. Oxidation of a compound means that it loses electron and and that addition of electron is reduction of a compound. When an organic compound is oxidized biologically, it usually loses electrons in the form of hydrogen atoms similarly, hydrogenation is the usual way of adding electron CH3 C O COOH Pyruvic acid + 2H (reduction of pyruvic acid) + 2H (oxidation of lactic acid) CH3 HC OH COOH Lactic acid NH2 N N O N N O H H H OH H H Nicotinamide adenine dinucleotide (NAD) HO P O O OH P O CONH2 N O O H H H OH H H Pairs of hydrogen atoms freed during oxidation or required in reductions are carried by nucleotide derivatives, especially nicotinamide adenine dinucleotide (NAD) and its phosphorylated form of NADP. NADH NAD+ H H HC HC H C N O C CH R Reduction form - 2H (oxidation) NH2 + 2H (reduction) HC HC C N O C CH R Oxidation form NH2 NAD serves two major functions 1. Analog to one of ATP’s job – reducing power made available during breakdown of nutrient is carried to biosynthetic reaction. The reducing power is used for the construction of cell components. When a metabolite is oxidized, NAD+ accepts two electrons plus a hydrogen ion (H+) and NADH results. NADH then carries energy to cell for other uses NAD and related pyridine nucleotide compounds carrying hydrogen also participate in ATP formation in aerobic metabolism. The hydrogen atoms in NADH are combined with oxygen in a cascade of reactions known as the respiratory chain. The energy released in this oxidation is sufficient to form three molecule of ATP from ADP. All the biological systems, e.g., anaerobic, aerobic, or photosynthetic metabolism, utilize ATP as central means of accumulating oxidative or radiant energy for driving the endergonic processes of the cell. CARBON CATABOLISM Breakdown of nutrients to obtain energy is called catabolism. Fermentation of carbohydrates, e.g., glucose, are under this category. The are at least seven glucose fermentation pathways and the particular one used and the end products produced depend on the microorganism involved Embden-Meyerhof-Parnas Pathway (EMP) Embden-Meyerhof-Parnas Pathway involved in ten enzyme catalyzed steps which start with glucose and end with pyruvate. The EMP steps involve isomerization, ring splitting, or transfer of a small group such as hydrogen or phosphate. Two moles of pyruvate are produced per mole of glucose passing through the pathway. ATP hydrolysis coupled with two reactions and each reaction involve sufficiently negative free negative energies to drive ADP phosphorylation. CH2OH H ATP O H OH ADP H H Hexokinase H OH CH2OPO32- H H OH OH H OH OH Glucose 6-phosphate Glucose CH2OH O Phosphohexoisomerase OH OH OH OH H H CH2OPO32O H H OH H Fructose 6-phosphate ATP Phosphof ructokinase ADP CH2OPO32- CH 2OPO32- HC O Triose isomerase HCOH CH 2OPO32Glyceraldehyde 3-phosphate C O CH 2OH Dihydroxyacetone phosphate CH2OPO32- O H Aldolase OH OH H OH H Fructose 1,6-diphosphate CH 2OPO 32HC O NAD+ HCOH CH 2OPO32- Glyceraldehyde 6-phosphate C OPO32Glyceraldehyde 3-phosphate dihydrogenase ATP ADP HCOH NADH 3-phosphoglycerate kinase O 1,3-Diphosphoglycerate CH 2OPO32HCOH COO3-Phosphoglycerate Phosphoglyceramutase H2 O CH 3 C O COOPyruvate ADP ATP Pyruvate kinase CH2 CH 2OH C O PO 32- HCOPO 32- COOPhosphoenolpyruvate Enolase COO2-Phosphoglycerate C6H12O6 + 2 Pi + 2 ADP + 2 NAD+ 2 C3H4O3 + 2 ATP + 2 (NADH + H+) Stored chemical energy and reducing power result from overall pathway. This is called substrate-level pathway In muscle cell and lactic acid bacteria, the reactions of the EMP are followed by single step C3H4O3 + NADH + H+ C3H6O3 + NAD+ The overall reaction sequence from glucose to lactic acid is called glycolysis Free-energy change for overall glycolysis reaction Glucose + 2 Pi + 2 ADP 2 lactose + 2 ATP + 2 H2O G0’ = -32,400 cal/mol With corresponding quantity for the glucose breakdown alone Glucose 2 lactose G0’ = -47,000 cal/mol A total free-energy of 14.6 kcal or 7.3 kcal for each mole of ATP generated has been conserved by the pathway as high energy phosphate compounds. Carbohydrate Catabolism The breakdown of carbohydrates to release energy – Glycolysis – Krebs cycle – Electron transport chain Other Carbohydrate Catabolic Pathways The pentose phosphate cycle or pathway begins by oxidizing glucose phosphate Glucose 6-phosphate + NADP+ 6-phosphogluconate + NADPH + H+ Major function of the pentose phosphate pathway is supplying the cell with NADPH which in turn carries electrons to biosynthetic reactions