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Introduction to Metabolic Biochemistry Basic Principles of Pathway Chemistry Metabolism is the mechanism of living cells to extract, convert and store energy from nutrients. Metabolism is a complex network of chemical reactions within the confines of a cell that can be analyzed in self-contained parts called pathways. Metabolic pathways contribute to catabolism - the oxidative degradation of molecules, and anabolism - the reductive synthesis of molecules. It is important to realize that pathways, be they catabolic or anabolic in nature, are interdependent and controlled (e.g. hormones) by the energy needs (e.g. growth, work, digestion) and physiological activity of an organism. The principals of metabolic pathways can be described by four conceptually distinct categories:
Homeostasis and Thermodynamics of Metabolic Pathways Metabolism is a largely circular process of energy conversion in cells of living organisms. Chemical energy is extracted from nutrients (catabolism) and this energy is in turn used to synthesize new molecules (anabolism) from the same type of nutrients to maintain the structure and function of an organism. To accomplish both, say energy extraction from and biosynthesis of proteins, metabolism of living cells is a spatial and temporal network of chemical reactions close to, but never at chemical equilibrium. Living organisms maintain a state of metabolic homeostasis which can be viewed as a steady-state throughput or flow of energy and metabolites to sustain body functions and structures. Nutrients provide both chemical energy and molecular building blocks to form macromolecules like proteins, DNA, polysaccharides, and membranes. These macromolecular structures in turn control nutrient uptake and metabolism. Along metabolic pathways the energy content of the initial substrate (glucose) differs significantly from the energy content of the product (carbon dioxide and water). In thermodynamic terms the change in Gibbs free energy, DG, is quite large (negative value). If such a reaction would occur at once, the chemical energy contained in sugar would be converted to heat. Enzymes of metabolic pathways are able to capture this energy in small portions and store it in form of internal high energy compounds drastically reducing the amount of energy lost as heat. Compare the caloric content of an ounce of bread to burning a small stick of wood (wood and starch contain the same energy-rich stuff - glucose). The heat of the flame represents the released energy. Converting glucose to carbon dioxide and water in one step has also one other important consequence; the reaction is irreversible due to a prohibitively large activation energy of converting carbon dioxide and water into sugar. Living organism have evolved metabolic pathways that allow at least partial reversibility of the conversion of such processes by providing many intermediate steps with small DG values close to zero, i.e., near their chemical equilibrium. Note that the serial conversion of metabolic intermediates allows the coupling of exergonic reactions with endergonic ones. This generally allows for a reversibility of pathways. Although some pathways are catalyzed by the same enzymes facilitating both directions for degradation and synthesis, most enzymes operated away from their chemical equilibrium and cells provide two separate, non-reversible pathways - one for degradation and one for biosynthesis. This allows for additional control by the cell to switch between biosynthesis of new macromolecules and the energy requirements to fuel those biosynthetic pathways, and also allows differences in metabolism and control of metabolism from cell type to cell type, or species to species.
Metabolic pathways follow a logic or concept that once understood, makes it easy to analyze and explore any pathway. The first step of a metabolic pathway normally includes the activation of the substrate into a high energy compound (also referred to as 'committed step'). Take as an example the first step in glycolysis (anaerobic glucose catabolism) where one molecule of glucose is phosphorylated before it can be used for oxidative catabolism: ATP + Glucose Û ADP + Glucose-6-P DG°' = -4.3kcal/mol (a) (DG -12kcal/mol) This reaction can be viewed as the sum of two separate reactions, a spontaneous one (exergonic) and one that requires energy (endergonic): ATP + H2O Û ADP + H3PO4 DG°' = -7.5kcal/mol (b; exergonic) Glucose + H3PO4 Û Glucose-6-P + H2O DG°' = +3.2kcal.mol (c; endergonic) The phosphorylation of glucose with inorganic phosphate costs +3.2kcal/mol and because of this large activation energy the reaction will not spontaneously occur in aqueous solution. ATP hydrolysis on the other hand is an exergonic reaction yielding -7.5kcal/mol. ATP (C00002) is indeed an unstable molecule in water and hydrolyzes spontaneously, albeit at a slow rate. The energy of this reaction is distributed as heat. Catalyzed by enzymes the energy of this reaction can be harnessed to power biosynthesis; here phosphorylation of glucose, an high energy compound. The use of high energy compounds in cellular metabolism High energy compounds power metabolic pathways. By definition high energy compounds are those with a standard free energy DG°' of more than 6kcal/mol). The most important and widely used high energy compound is ATP and the related nucleotides CTP, UTP, and GTP. The major source of ATP comes from respiration (mitochondrial process depend on molecular oxygen) and photosynthesis. If oxygen or light are not readily available, other high energy compounds serve as emergency fuel to restore ATP levels. These are the so called phosphagens like phosphocreatine in vertebrates and phosphoarginine in invertebrates. Except for ATP which serves as a multipurpose energy donor, most high energy compounds are used for specific metabolic pathways only (but never only one). Cytosine triphosphate (CTP) is used in phospholipid synthesis by generating activated intermediates (e.g. CDP-choline). Uridine triphosphates are used for complex carbohydrate synthesis (glycogen, cellulose) by generating activated sugars (e.g. UDP-glucose). Guanosine triphosphate is important in protein synthesis. Phosphocreatine is used mainly in skeletal muscle to provide chemical energy for intense work and assure a constant supply of ATP. The enzyme creatine kinase (EC 2.7.3.2) catalyzes the transfer of the phosphate group between the two metabolites. Because the change in free energy of ATP regeneration from ADP and phosphocreatine is only slightly negative, the reaction is readily reversible and mainly dominated by the equilibrium concentration of the reactants. An other important activated chemical group besides phosphate ester bonds are the acyl thioesters (acyl-CoA) of short chain compounds (acetyl-CoA) and long-chain fatty acids for lipid metabolism. Numerous other high-energy intermediates are activated in order to promote group transfer reactions. Their formation is directly or indirectly dependent on the energy available from ATP. The Importance of Enzymes Glucose phosphorylation does not happen spontaneously by simply mixing glucose with ATP in aqueous solution. The coupling of an energy yielding reaction with an energy consuming one is done by enzymes. Often enzymes catalyze the two reactions in series by providing a structural scaffold that optimally orients the two reactants (substrates) to promote a group transfer reaction from donor X to acceptor Y. The enzyme may serve as an intermediary binding site of the transferred group. Importantly, while the two steps (b and c) shown above each depend on water as co-substrate, no H2O is involved in the enzyme mediated coupled process (reaction a). Enzyme also provide exquisite substrate specificity. In liver, glucose phosphorylation, the transfer of an high energy phosphate bond from ATP to glucose is catalyzed by hexokinase 2 (E.C. 2.7.1.1 for human isoform; or glucokinase EC 2.7.1.2 for invertebrates and microorganisms). Oxidation-Reduction Reactions All energy yielding process are ultimately dependent upon enzymatically catalyzed redox reactions. The most important one for energy metabolism involve biological membranes with bound electron transport processes like photosynthesis and oxidative phosphorylation. Biological oxidation is the primary provider of energy for cellular anabolism, the reductive synthesis of metabolites, by furnishing mobile hydrogens, and phosporylating energy by combining hydrogens with oxygen to form water coupling this process to the production of ATP in the form of oxidative phosphorylation. Central to the oxidation-reduction processes are the vitamin B group containing coenzymes nicotinamide-adenine dinucleotide (NAD) and nicotinamide-adenine dinucleotide phosphate (NADP, (C00006; oxidized form); NAD (C00003; oxidized form; not phosphorylated at the adenosine ribosyl C2 position). Being part of the appropriate enzymes the oxidized nicotinamide ring of NAD+ or NADP+ extracts a hydride (H:-) from a wide variety of simple metabolites in a process known as dehydrogenation. The enzymes catalyzing the reduction of nicotinamide containing coenzymes are called dehydrogenases. In a typical reaction two hydrogen atoms (including their electrons) are removed from the substrate to produce the oxidized form of the donor. The fate of the two hydrogens differs: one hydrogen with two electrons (H:-), a hydride ion, is transferred to the nicotinamide ring to produce reduced NADH or NADPH while the other hydrogen is released into solution as a free proton (H+). The generic form of a redox reaction mechanism catalyzed by enzymes with NAD as cofactor is shown. The use of these nicotinamide based redox reactions provides versatility and reversibility. Under most cellular conditions, the free energy change is small and dehydrogenases catalyze both oxidative and reductive reactions. Many different types of substrates are used as partners for NAD and NADP - carbohydrates, lipids, and amino acids. The coenzymes are diffusible and facilitate the shuttling of hydrogen atoms and electrons among different dehydrogenases that belong to different pathways. The different phosphorylation state of NAD and NADP provides a control mechanism to use the respective coenzymes for different classes of pathways. The phosphorylation does not affect the redox potential of the coenzymes (see below), but the affinity of the molecules for specific proteins. The example of a redox reaction discussed here is the anaerobic regeneration of NAD+ reduced during glycolysis by the reduction of pyruvate to lactate. NADH + H+ + Pyruvate Û NAD+ + Lactate This reaction is catalyzed by lactate dehydrogenase (human enzyme E.C. 1.1.1.27). Lactate dehydrogenase couples the two half-reactions:
NAD+ + 2H+ + 2e- Û
NADH + H+
Eo' = -0.32V The standard reduction potential Eo' is used to quantify redox reactions instead of the change of standard free energy. A negative reduction potential indicates a reduction reaction, i.e., the binding of two electrons (and two concomitant H+). The half-reaction with the more negative Eo' will act as electron donor or reducing agent. The metabolically more important regeneration of NAD+ is the aerobic (oxygen dependent) process of using the reductive power of NADH for the synthesis of ATP (note that positive Eo' values indicate spontaneous reactions). NADH + H+ + ½O2 Û NAD+ + H2O Eo' = +1.14V This process is strictly dependent on the presence of molecular oxygen and is called oxidative phosphorylation. During the oxidation of glucose to carbon dioxide and water, most of the reducing power is not generated by glycolysis (in the cytoplasm of the cell), but the tricarboxylic acid cycle, also known as Citric Acid or Krebs cycle (in the cell's mitochondria). The complete aerobic oxidation of one molecule of glucose yields 30-36 molecules of ATP (only 2 come from glycolysis), of which 90% is captured in the form of reducing power and converted to ATP through oxidative phosphorylation in the mitochondrial inner membrane. In the process, six units of carbon dioxide and six water molecules are generated. Glucose (C6H12O6) Û 6CO2 + 6H2O ~ 30-36 ATP Again, the chemical energy of the covalent bond structure of glucose is captured in small steps involving many metabolic intermediates rendering the process partially reversible, also the overall process is not. Humans can regenerate glucose from the metabolic intermediate pyruvate but not CO2. Humans as we all know are not able to synthesize glucose from 'scratch', i.e., carbon dioxide, a process known as photosynthesis in plants and some microorganisms. Respiration and photosynthesis are catalytically possible only
because of the coordinated activity of hundreds of proteins that
belong to deferent sets of pathways in different compartments of
cells and/or organisms. Understanding the structural and functional
complexity that provides reductive synthesis of glucose as well
as oxidative degradation is the same as understanding the mechanism
of cellular metabolism. Metabolic pathways can be controlled at three levels. First, the amount of enzymes available through transcription (gene expression), translation, and protein turnover (proteolytic degradation). Second, the catalytic activity of an enzyme is controlled by allosteric modulators, activators and competitive inhibitors (agonists and antagonists, respectively), and post-translational modifications such as phosphorylation, acetylation, and glycosylation under the control of hormones, growth-factors, and neurotransmitters. Third, substrate availability (concentration) is controlled by steady-state equilibrium and compartmentalization. The latter depends on active membrane transport systems (pumps, transporters; chemical energy required) and the passive diffusion via substrate specific membrane proteins (ion channels, facilitators; no chemical energy required; diffusion is controlled by concentration gradients). H
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