Metabolism overview

Recommended questions from chapter 17: 1,4,5,6,7,8,9,10,13,15,16,17,18,19.

Metabolism consists of two processes:

1. Catabolism is the oxidative degradation of of complex nutrients into simple molecules. These reactions are performed to generate free energy or utilize the molecular components that make up the nutrients. For the most part, catabolism is oxidative, and electrons are transferred to the coenzymes NAD+ and NADP+. NADH is used in additional ATP generation reactions. NADPH is used as an electron donor in biosynthesis reactions, which are reductive.

2. Anabolism is the synthesis of required molecules from simple components. NADPH is a commonly used electron donor for the reductive anabolic reactions, and ATP is commonly used as a free energy source for these reactions.

NAD+ is used as an electron acceptor in oxidative catabolism

The general form of this hydride transfer reaction is

+ +. AH2 + NAD A + NADH + H

The reaction shown is catalyzed by alcohol dehydrogenase. NADPH has a phosphate at the 2’ position of the adenosine.

1 Reduction of FAD to FADH2

Flavin (oxidized form, quinone, e.g., FAD) radical form, semiquinone, e.g., FADH

radical form, semiquinone reduced form, hydroquinone, e.g., FADH2

Oxidation of nutrients is carried out by all organisms. Even obligate anaerobes (which do not carry our oxidative phosphorylation) oxidize substrates to drive ATP synthesis.

The two most widely occurring electron carriers are Nicotinamide adenine dinucleotide (NAD), and Flavin adenine dinucleotide (FAD).

Metabolic strategies

1. Autotrophs (auto = self, trophos = feeder) can generate all their requirements from simple molecules such as H2O, CO2, NH3, and H2S.

A. Chemilothotrophs (litho = stone) obtain free energy by oxidation of inorganic 2 compounds such as NH3, H2S, or Fe +:

H2S + 4O2 → H2SO4 2NH3 + 4O2 → 2HNO3 + 2H2O 4FeCO3 + O2 + 6H2O → 4Fe(OH)3 + 4CO2

B. Phototrophs obtain free energy from sunlight. Transfer of electrons from donors such as H2O is used to power the production of carbohydrates (CH2O)n, which are then oxidized on demand to release free energy.

2. Heterotrophs (hetero = other) obtain free energy through consumption and oxidation of organic compounds (proteins, lipids, carbohydrates).

A. Obligate aerobes must use O2 for oxidation, so they (us!) depend on O2 consumption.

B. Obligate anaerobes cannot use O2, which is a poison for these organisms. Instead, they use nitrate or sulfate as oxidizing agents (halophilic bacteria). C. Facultative anaerobes can grow in the presence or absence of O2 (yeast, E. coli).

2 Metabolic pathways •Metabolic pathways are a series of reactions that are normally catalyzed by enzymes. •There are over 2000 reactions known for the (relatively) simple organism E. coli. •These reactions share intermediates and products so the study of one reaction in isolation is somewhat arbitrary. •In general, catabolic reactions involve oxidizing metabolites in a process that yields free energy (exergonic). The metabolite is broken down into a 2-carbon product that is covalently linked to coenzyme A, to yield acetyl-CoA. The energy released in this process is utilized to synthesize ATP + from ADP and Pi , reduce NAD to NADH, or reduce FAD to FADH2. •A few intermediates are shared in the catabolism of many substrates. This process is followed by the oxidation of acetyl carbons to CO2 (citric acid cycle) and reduction of NAD+ to NADH. Oxidation of NADH and

FADH2 using oxidative phosphorylation results in the production of ATP.

Thermodynamic considerations in metabolism

1. Near equilibrium reactions.

For the process A + B ↔ C + D the free energy change, ∆G, is related to the standard free energy change (∆G°’) and the concentrations of the reactants:

[C][D] ΔG = ΔGo'+RTln( ) [A][B]

If the concentrations of reactants and products are close to their equilibrium values, then ([C][D])/([A][B]) ≈ Keq. Because, by definition -RTln(Keq) = ∆G°’, then ∆G = 0 when the reactants and€ products are near their equilibrium values.

These types of reactions are called “near equilibrium” reactions, and their forwards and reverse rates can be easily regulated by changing the concentrations of reactants and products.

3 Thermodynamic considerations in metabolism

2. “Far from equilibrium” reactions.

Some metabolic reactions lie far from equilibrium, so the reaction is irreversible. This is the case when the free energy of the products is much lower than the free energy of the reactants. Here the reverse reaction becomes very unlikely, so the overall reaction, for the most part, is irreversible, even in the presence of a catalyst.

In these reactions ∆G << 0 and a change in the concentration of reactant has little effect on the reaction rate. The enzyme is essentially saturated, so the reaction is almost 0-order with respect to substrate.

Changes in the activity of the enzyme, through, for example, allosteric regulation, can change the rate of the reaction.

Thermodynamic considerations in metabolism •The flux (flow rate) of metabolites through the metabolic pathway can be understood if the enzymes catalyzing the reactions of the pathway are understood in terms of reaction reversibility. •Most enzymes operate at near-equilibrium conditions, such that variations in the substrate and product concentrations alter reaction rates. •Some reactions within the pathway, however, are irreversible.

There are several implications for this situation:

1. Metabolic pathways are irreversible. If one step in the pathway is irreversible, the entire pathway is irreversible. This means that metabolic pathways are directional.

2. Metabolic pathways have an early “committing” step. Often, the irreversible step is at the beginning of the pathway. This means that the reactants are “committed” to the pathway.

3. Catabolic and anabolic pathways differ. The synthesis and degradation pathways are NOT the simple reversal of each other. If substance 2 is needed, it is necessary to turn off pathway B and turn on pathway A. This level of control would not be possible if pathway A was the simple reversal of pathway B.

4 Regulation of metabolic pathways

•The flux (flow rate) of nutrients through an organism (an open thermodynamic system) is relatively constant. This means that an organism maintains the concentration of metabolic pathway intermediates constant. •The net flux within a system can be described by J = vf - vr, and at equilibrium, by definition, there is no net flux. A state of equilibrium is inconsistent with life. •The rate-limiting step (often the first committing step in the pathway) is by definition the slowest step in the pathway. In such cases the rate is not affected by substrate concentrations because the enzyme is saturated, so this step is far from equilibrium. The product of the rate- limiting step is removed by successive steps that are carried out by enzymes at near-equilibrium conditions. • To alter the rate-liming step, several strategies are utilized:

1. Allosteric control: the product of the pathway has an inhibiting effect on the rate-liming step. For example, the inhibition of aspartate transcarbamoylase by CTP.

2. Covalent modification of the enzyme that carries out the rate-limiting step. For example, by a kinase/phosphorylase reaction.

3. Independent pathways for catabolism and anabolism.

4. Genetic control over the level of the rate-limiting enzyme.

“High energy” compounds in metabolism

ATP is a common “energy currency” which is used to store the free energy released by catabolic processes.

∆Gº’ of ATP hydrolysis is -30.5 kJ/mole. Under cellular concentration of ADP, Pi and ATP, the ∆G of ATP hydrolysis is much greater because the concentrations of ADP, Pi, and ATP are much lower than 1M. Remember that ∆G for ATP hydrolysis is given by

[ADP][P ] ΔG = ΔGo'+RTln( i ) [ATP]

If the concentration of each of these reagents is 2 mM, what is ∆G? Why is ATP a convenient€ “energy currency?” What is the usefulness of a “high-energy” compound?

5 Why are the β and γ bonds in ATP “high-energy” bonds?

Remember not to confuse “high energy bond” with “bond energy!”

The phosphoanhydride bond requires high energy to form (and releases high energy when hydrolyzed) because of:

1. Resonance stabilization of the product.

2. Electrostatic repulsions between the changed phosphoanhydride groups.

3. In aqueous solutions, it is easier to solvate the hydrolysis products than the unreacted groups.

Other “high-energy” phosphate compounds If ATP is consumed very quickly, other “high energy” compounds can serve as “backup” molecules that can replenish the ATP concentration if it dips too low. An average person at rest consumes and regenerates ATP at ~ 3 moles/hr (~1.5 kg/hr) at rest and as much as 10 fold faster with exercise.

Most likely, acyl phosphate hydrolysis is driven by the same energetic considerations as ATP.

O OH O

2- 2- 2- H3C C OPO3 O3P C C C OPO3 H2 H

Acetyl phosphate 1,3-Bisphosphoglycerate

Glycerol-3 phosphate and glucose-6- phosphate don’t exhibit the same hydrolysis product stabilization of ATP, and their ∆Gº’ is in fact lower than that of ATP.

OH

2- HO C C C OPO3 H2 H2

H

Glycerol-3-phosphate

6 Thioesters

•Thioester is a candidate for a “primitive” high-energy compound.

•Thioesters are found in the central metabolic pathways of all known organisms. Thioesters were, presumably, more common in the pre- biotic world than phosphate compounds. Phosphate is less abundant in non living systems than thioesters.

•In metabolic pathways the thioester appears as a part of acetyl-coenzyme A (CoA). This molecule is an intermediate in carbohydrate, fatty acid, and amino acid catabolism. ∆Gº’ of acetyl-CoA hydrolysis is -31.5 kJ/mol.

The Nerst equation and reduction potential Electrons that are transferred from one group to another can be accounted for by an electro- chemical cell, where each half reaction is carried out. If n is the number of electrons transferred from reductant B to oxidant A (A is reduced), then in general form n + n + Aox + Bred ↔ Ared + Box

The free energy of this reaction is given by

€ n + 0 [Ared ][Box ] ΔG = ΔG + RTln( n + [Aox ][Bred ]

€

7 The Nerst equation and reduction potential

∆G is the amount of electrical energy required to transfer n moles of electrons across a potential difference ∆E, which is given in volts (1V = J/C). This can be expressed as

Δ G = −nFΔE where F is the faraday constant, which is the electrical charge of 1 mole of electrons, -1 -1 -1 96494 C•mol = 96494 J•V€• mol , and n is the number of electrons transferred per mole of reactants.

The Nernst equation describes the reduction potential, ∆E : RT [A ][Bn + ] ΔE = ΔE 0 − ln( red ox ) n [An + ][B ] F ox red

Where ∆E0 is the reduction potential when all the components are in their standard state. A positive ∆E yields€ a negative ∆G, which denotes a favorable reaction.

Experimental approaches in metabolic studies What we want to know: 1. The sequence of reactions by which a metabolite is converted to end product, and the energetics of these reactions. 2. The mechanism of each reaction step. 3. The regulation of each reaction step.

Approaches: 1. Trace metabolic labeling. A. Chemical labeling can be used. Disadvantage = chemical differences between labeled and unlabeled reactants. B. Isotopic labeling. Chemically identical to the unlabeled species. Disadvantages include radioactive waste disposal problems with the long-lived isotopes, such as 3H and 14C, as well as ethical issues that can be associated with such strategies.

For example, in 1945 David Shemin and David Rittenberg asked what nutrient was the source of the nitrogen in the heme group. They fed rats [15N]nutrients, isolated the heme group, and analyzed the heme group for the presence of 15N. Only when [15N]glycine was fed to the rats was the heme group labeled.

NMR can be used to detect isotopes such as 3H, 2H, 13C, and 31P, which is useful in studying phosphate-containing compounds. This can be done in vivo.

8 Determining precursor-product relationship Sometimes the reaction products and reactants are very similar and it is difficult to identify which is the product and which is the reactant. For example, oxidation-reduction products and reactants can interconvert in vitro (and in vivo), so both become labeled, which confounds the issue of which precedes which in the pathway. An approach that is used often is a pulse-chase experiment. A labeled known precursor is administered, then unlabeled precursor is added in excess (100 to 10000 fold), and the label is followed over time.

Consider the pathway:

[A*] A B end product → → [B*]

Criteria: 1. The peak of radioactivity in A must appear before the peak in B.

2. While the radioactivity in the product specific activity is rising, it must be less than that of the precursor. 3. After the radioactivity of the product has peaked, it must be greater than the 0 2 4 6 8 10 12 14 16 18 20 radioactivity of the precursor. time (min)

Perturbation of the pathway

If an enzyme inhibitor is found, one can use this inhibitor to find out the precursor the enzyme may act on within a putative pathway. For A → B → C →D, if the enzyme that catalyzes C → D is inhibited, C will accumulate. This is a way of showing that the enzyme is involved in this pathway. Under what conditions could this approach be misleading?

DNA expression arrays and proteomics

The DNA array approach is used to measure RNA levels in cells under difference conditions. Proteomics approach measures the amounts of protein levels in cells under different metabolic conditions.

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