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 (1)  We can write (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)  where  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) (4)

 Consider the reaction between two isomers in the Embden-Mayerhof pathway for glucose breakdown  Where P denotes phosphate. Because of the negative free energy change, equilibrium favors the dihydroxyacetone by a 22:1 ratio.  G 0 ’= cal/mol

 Many biological reaction and energy conversion process involve oxidation-reduction reaction such as  This type of reaction is described using the standard potential change where is the standard half-cell potential for the half reaction

 As a reference point for half-cell potential value, the hydrogen half-cell (at pH=0) is assigned a value of zero:  The free energy change and corresponding potential changes are related by  Where n is the number of electrons transferred and F is equal to kcal/V mol

Metabolic Reaction Coupling: ATP and NAD

ATP  Energy is released as food is oxidized  Used to form ATP from ADP and P i ADP + P i + Energy ATP  In cells, energy is provided by the hydrolysis of ATP ATP ADP + P i + Energy

 The enzymatic hydrolysis of ATP to yield ADP and inorganic phosphate has a large negative free- energy change Where P i 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 ATP + H 2 O  ADP + P i  G 0 ’ = -7.3 kcal/mol

 Embden-Meyerhof-Parnas pathway serves to illustrate the concept of a common chemical intermediate 1.Oxidation of aldehyde to carboxylic acid 2.Same reactions, coupled to ATP generation (glucose oxidation)

3.Reaction 2 and 1 yield

 Example

 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  G 0 ’ for ATP hydrolysis. Example, phosphoenolpyruvate  G 0 ’ = kcal mol -1. 1,3-diphosphoglycerate  G 0 ’ = kcal mol -1 1,3-diphosphoglycerate  G 0 ’ = 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  G 0 ’ = -3.3 kcal mol -1 glycerol-1-phosphate  G 0 ’ = -2.2 kcal mol -1 glycerol-1-phosphate  G 0 ’ = -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

Nicotinamide adenine dinucleotide (NAD)

 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 + Reduction formOxidation form

 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. Glucose Glucose 6-phosphateFructose 6-phosphate Fructose 1,6-diphosphate Dihydroxyacetone phosphate Glyceraldehyde 3-phosphate Triose isomerase Aldolase

Glyceraldehyde 6-phosphate 3-Phosphoglycerate1,3-Diphosphoglycerate 2-PhosphoglyceratePhosphoenolpyruvatePyruvate

C 6 H 12 O P i + 2 ADP + 2 NAD + 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 The overall reaction sequence from glucose to lactic acid is called glycolysis C 3 H 4 O 3 + NADH + H + C 3 H 6 O 3 + NAD + 2 C 3 H 4 O ATP + 2 (NADH + H + )

 Free-energy change for overall glycolysis reaction  With corresponding quantity for the glucose breakdown alone  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. Glucose + 2 Pi + 2 ADP 2 lactose + 2 ATP + 2 H 2 O  G 0 ’ = -32,400 cal/mol Glucose 2 lactose  G 0 ’ = -47,000 cal/mol

 The breakdown of carbohydrates to release energy –Glycolysis –Krebs cycle –Electron transport chain Carbohydrate Catabolism

Other Carbohydrate Catabolic Pathways  The pentose phosphate cycle or pathway begins by oxidizing glucose phosphate Major function of the pentose phosphate pathway is supplying the cell with NADPH which in turn carries electrons to biosynthetic reactions Glucose 6-phosphate + NADP + 6-phosphogluconate + NADPH + H +