RAMACHANDRAN PLOT:
The bond angles resulting from rotations at Cα are labeled ɸ (phi) for the N --- Cα bond and Ψ (psi) for the C---Cα bond.
By convention, both ɸ and Ψ are defined as 180◦when the polypeptide is in its fully extended conformation and all peptide groups are in the same plane.
In principle, ɸ and Ψ can have any value between -180 and +180◦ but many values are prohibited by steric interference between atoms in the polypeptide backbone and amino acid side chains.
The conformation in which both ɸ and Ψ are 0◦ is prohibited for this reason; this conformation is used merely as a reference point for describing the angles of rotation.
Values for ɸ and Ψ are graphically revealed when Ψ is plotted versus ɸ in a Ramachandran plot.
The range for Pro residues is greatly restricted because ɸ is limited by the cyclic side chain to the range of -35◦ to -85◦
Proline also disfavors α helix formation because it lacks an amide H atom for hydrogen bonding.
BIOENERGETICS AND METABOLISM:
TERMINOLOGY
Catabolism is the degradative phase of metabolism in which organic nutrient molecules (carbohydrates, fats, and proteins) are converted into smaller, simpler end products (such as lactic acid, CO2, NH3).
Catabolic pathways release energy, some of which is conserved in the formation of ATP and reduced electron carriers (NADH, NADPH, and FADH2); the rest is lost as heat.
In anabolism, also called biosynthesis, small, simple precursors are built up into larger and more complex molecules, including lipids, polysaccharides, proteins, and nucleic acids.
Anabolic reactions require an input of energy, generally in the form of the phosphoryl group transfer potential of ATP and the reducing power of NADH, NADPH, and FADH2.
Some processes can be either catabolic or anabolic, depending on the energy conditions in the cell. These are referred to as amphibolism.
The term exergonic is used to denote a chemical reaction which liberates chemical-free energy. The term exothermic refers to the total energy liberated including heat.
The corresponding energy-consuming term endergonic refers to the processes which require an input of free energy while the term endothermic denotes a total energy requirement including heat.
Five main reaction classes you will encounter in upcoming chapters.
Most of the reactions in living cells fall into one of five general categories:
(1) Oxidation-reductions;
(2) Reactions that make or break carbon–carbon bonds;
(3) Internal rearrangements, isomerizations, and eliminations
(4) Group transfers
(5) free radical reactions.
Oxidation-reduction reactions
Carbon atoms encountered in biochemistry can exist in five oxidation states, depending on the elements with which carbon shares electrons,
Biological oxidation, a carbon atom becomes covalently bonded to an oxygen atom.
The enzymes that catalyze these oxidations are generally called oxidases or, if the oxygen atom is derived directly from molecular oxygen (O2), oxygenases.
Oxidation reactions generally release energy.
Most living cells obtain the energy needed for cellular work by oxidizing metabolic fuels such as carbohydrates or fat;
The high affinity of O2 for electrons makes the overall electron-transfer process highly exergonic, providing the energy that drives ATP synthesis—the central goal of catabolism.
5 oxidation state of carbon
Reactions that make or break carbon–carbon bonds
Heterolytic cleavage of a COC bond yields a carbanion and a carbocation.
Carbon of a carbonyl group has a partial positive charge due to the electron-withdrawing nature of the adjacent bonded oxygen, and thus is an electrophilic carbon.
The presence of a carbonyl group can also facilitate the formation of a carbanion on an adjoining carbon, because the carbonyl group can delocalize electrons through resonance.
The importance of a carbonyl group is evident in three major classes of reactions in which C-C bonds are formed or broken
Aldol condensations (such as the aldolase reaction;
Claisen condensations (as in the citrate synthase reaction, and
Decarboxylations (as in the acetoacetate decarboxylase reaction
For both the aldol condensation and the Claisen condensation, a carbanion serves as nucleophile and the carbon of a carbonyl group serves as electrophile.
The carbanion is stabilized in each case by another carbonyl at the carbon adjoining the carbanion carbon
Internal rearrangements, isomerizations, and eliminations
In which redistribution of electrons results in isomerization, transposition of double bonds, or cis-trans rearrangements of double bonds.
formation of fructose 6-phosphate from glucose 6-phosphate during sugar metabolism
Group transfer reactions
The transfer of acyl, glycosyl, and phosphoryl groups from one nucleophile to another is common in living cells.
Acyl group transfer generally involves the addition of a nucleophile to the carbonyl carbon of an acyl group to form a tetrahedral intermediate.
Nucleophilic substitution is made more favorable by the attachment of a phosphoryl group to an otherwise poor leaving group such as -OH.
Phosphorus can form five covalent bonds. The conventional representation of Pi with three P-O bonds and one P=O bond, is not an accurate picture.
In Pi, four equivalent phosphorus–oxygen bonds share some double-bond character, and the anion has a tetrahedral structure . As oxygen is more electronegative than phosphorus, the sharing of electrons is unequal: the central phosphorus bears a partial positive charge and can therefore act as an electrophile.
Hexokinase, for example, “moves” a phosphoryl group from ATP to glucose.
Free radical reactions
Once thought to be rare, the homolytic cleavage of covalent bonds to generate free radicals has now been found in a range of biochemical processes. Some examples are the reactions of methylmalonyl- CoA mutase , ribonucleotide Reductase and DNA photolyase
PRINCIPLES OF BIOENERGETICS::
Cells Require Sources of Free Energy
Cells are isothermal systems—they function at essentially constant temperature (they also function at constant pressure).
Heat flow is not a source of energy for cells, because heat can do work only as it passes to a zone or object at a lower temperature.
Heterotrophic cells acquire free energy from nutrient molecules, and photosynthetic cells acquire it from absorbed solar radiation. Both kinds of cells transform this free energy into ATP.
Gibbs free energy ∆G
The free-energy change, ∆G, has a negative value and the reaction is said to be exergonic/spontaneous. In endergonic reactions, the system gains free energy and ∆G is positive.
The second law of thermodynamics states that a process can occur spontaneously only if the sum of the entropies of the system and its surroundings increases. This can be represented as:
Thus, the total entropy of a system must increase if a process is to occur spontaneously. However, the entropy of a system can decrease even during a spontaneous process, provided the entropy of the surroundings increases to such extent that their sum is positive.
The Standard Free-Energy Change Is Directly Related to the Equilibrium Constant
The complete oxidation of organic compounds such as glucose or palmitate to CO2 and H2O, which in cells requires many steps, results in very large decreases in standard free energy.
Actual Free-Energy Changes Depend on Reactant and Product Concentrations:
We must be careful to distinguish between two different quantities: the free-energy change, ∆G, and the standard free-energy change, ∆G◦
The standard free energy change tells us in which direction and how far a given reaction must go to reach equilibrium when the initial concentration of each component is 1.0 M, the pH is 7.0, the temperature is 25 ◦C, and the pressure is 101.3 kPa. Thus ∆G◦ is a constant: it has a characteristic, unchanging value for a given reaction. But the actual free-energy change, _G, is a function of reactant and product concentrations.
The value of ∆G provides no information about the rate of a reaction. A negative ∆G indicates that a reaction can occur spontaneously, but it does not signify that it will occur at a perceptible rate.
.
The concentration terms in this equation express the effects commonly called mass action, and the term [C][D]/[A][B] is called the mass-action ratio, Q.
The criterion for spontaneity of a reaction is the value of ∆G, not ∆G◦. A reaction with a positive ∆G◦ can go in the forward direction if ∆G is negative.
The free-energy change for a reaction is independent of the pathway by which the reaction occurs; it depends only on the nature and concentration of the initial reactants and the final products. Enzymes cannot, therefore, change equilibrium constants; but they can and do increase the rate at which a reaction proceeds in the direction dictated by thermodynamics.
Standard Free-Energy Changes Are Additive
Phosphoryl Group Transfers and ATP::
ATP as the energy currency that links catabolism and anabolism.
Heterotrophic cells obtain free energy in a chemical form by the catabolism of nutrient molecules, and they use that energy to make ATP from ADP and Pi. most cases of energy donation by ATP involve group transfer, not simple hydrolysis of ATP.
The Free-Energy Change for ATP Hydrolysis Is Large and Negative:
The hydrolytic cleavage of the terminal phosphoric acid anhydride (phosphoanhydride) bond in ATP separates one of the three negatively charged phosphates and thus relieves some of the electrostatic repulsion in ATP;
Although the hydrolysis of ATP is highly exergonic (∆G◦=-30.5 kJ/mol), the molecule is kinetically stable at pH 7 because the activation energy for ATP hydrolysis is relatively high. Rapid cleavage of the phosphoanhydride bonds occurs only when catalyzed by an enzyme.
Mg2- in the cytosol binds to ATP and ADP, and for most enzymatic reactions that involve ATP as phosphoryl group donor, the true substrate is MgATP2-.
Other Phosphorylated Compounds and Thioesters Also Have Large Free Energies of Hydrolysis:
Hydrolysis of phosphoenolpyruvate (PEP).
Hydrolysis of phosphocreatine.
In all these phosphate-releasing reactions, the several resonance forms available to Pi stabilize this product relative to the reactant, contributing to an already negative free-energy change.
Thioesters, in which a sulfur atom replaces the usual oxygen in the ester bond, also have large, negative, standard free energies of hydrolysis.
Acetyl-coenzyme A, or acetyl-CoA, is one of many thioesters important in metabolism. The acyl group in these compounds is activated for transacylation, condensation, or oxidation-reduction reactions.
Thioesters undergo much less resonance stabilization than do oxygen esters; consequently, the difference in free energy between the reactant and its hydrolysis products, which are resonance-stabilized, is greater for thioesters than for comparable oxygen esters.
In both cases, hydrolysis of the ester generates a carboxylic acid, which can ionize and assume several resonance forms.
Together, these factors result in the large, negative ∆G (-31 kJ/mol) for acetyl-CoA hydrolysis.
for hydrolysis reactions with large, negative, standard free-energy changes, the products are more stable than the reactants for one or more of the following reasons:
(1) The bond strain in reactants due to electrostatic repulsion is relieved by charge separation, as for ATP.
(2) The products are stabilized by ionization, as for ATP, acyl phosphates, and thioesters.
(3) The products are stabilized by isomerization (tautomerization), as for phosphoenolpyruvate.
(4) The products are stabilized by resonance.
ATP Provides Energy by Group Transfers, Not by Simple Hydrolysis:
ATP hydrolysis per se usually accomplishes nothing but the liberation of heat, which cannot drive a chemical process in an isothermal system.
The energy-dependent reactions catalyzed by helicases, RecA protein, and some topoisomerases also involve direct hydrolysis of phosphoanhydride bonds.
GTP-binding proteins that act in signaling pathways directly hydrolyze GTP to drive conformational changes that terminate signals triggered by hormones or by other extracellular factors.
“High-energy” compounds have a ∆G◦ of hydrolysis more negative than -25 kJ/mol; “low-energy” compounds have a less negative ∆G◦.
The term “high-energy phosphate bond,” long used by biochemists to describe the P-O bond broken in hydrolysis reactions, is incorrect and misleading as it wrongly suggests that the bond itself contains the energy. In fact, the breaking of all chemical bonds requires an input of energy.
The free energy released by hydrolysis of phosphate compounds does not come from the specific bond that is broken; it results from the products of the reaction having a lower free-energy content than the reactants.
The transfer of a phosphoryl group to a compound effectively puts free energy into that compound, so that it has more free energy to give up during subsequent metabolic transformations.
One more chemical feature of ATP is crucial to its role in metabolism: although in aqueous solution ATP is thermodynamically unstable and is therefore a good phosphoryl group donor,
ATP is kinetically stable. Because of the huge activation energies (200 to 400 kJ/mol) required for uncatalyzed cleavage of its phosphoanhydride bonds, ATP does not spontaneously donate phosphoryl groups to water or to the hundreds of other potential acceptors in the cell. Only when specific enzymes are present to lower the energy of activation does phosphoryl group transfer from ATP proceed.
ATP Donates Phosphoryl, Pyrophosphoryl, and Adenylyl Groups:
ATP is an energy-donor substance or a coenzyme
The reactions of ATP are generally SN2 nucleophilic displacements, in which the nucleophile may be, for example, the oxygen of an alcohol or carboxylate, or a nitrogen of creatine or of the side chain of arginine or histidine. Each of the three phosphates of ATP is susceptible to nucleophilic attack, and each position of attack yields a different type of product.
Notice that hydrolysis of the α–β phosphoanhydride bond releases considerably more energy (~46 kJ/mol) than hydrolysis of the β–γ bond (~31 kJ/mol)
Nucleophilic attack at the α position of ATP displaces PPi and transfers adenylate (5-AMP) as an adenylyl group the reaction is an adenylylation.
The PPi formed as a byproduct of the adenylylation is hydrolyzed to two Pi by the ubiquitous enzyme inorganic pyrophosphatase, releasing 19 kJ/mol and thereby providing a further energy “push” for the adenylylation reaction. In effect, both phosphoanhydride bonds of ATP are split in the overall reaction. Adenylylation reactions are therefore thermodynamically very favorable.
When the energy of ATP is used to drive a particularly unfavorable metabolic reaction, adenylylation is often the mechanism of energy coupling. Fatty acid activation is a good example of this energy-coupling strategy.
Transphosphorylations between Nucleotides Occur in All Cell Types:
Several enzymes then carry phosphoryl groups from ATP to the other nucleotides.
Nucleoside diphosphate kinase, found in all cells, catalyzes the reaction
The enzyme actually catalyzes a two-step phosphoryl transfer, which is a classic case of a double-displacement (Ping-Pong) mechanism .
First, phosphoryl group transfer from ATP to an active-site His residue produces a phosphoenzyme intermediate; then the phosphoryl group is transferred from the P –His residue to an NDP acceptor.
During periods of intense demand for ATP, the cell lowers the ADP concentration, and at the same time acquires ATP, by the action of adenylate kinase:
This reaction is fully reversible, so after the intense demand for ATP ends, the enzyme can recycle AMP by converting it to ADP, which can then be phosphorylated to ATP in mitochondria.
A similar enzyme, guanylate kinase, converts GMP to GDP at the expense of ATP.
Phosphocreatine , also called creatine phosphate, serves as a ready source of phosphoryl groups for the quick synthesis of ATP from ADP.
The enzyme creatine kinase catalyzes the reversible reaction
Organisms in the lower phyla employ other PCr-like molecules (collectively called phosphagens) as phosphoryl reservoirs.
Inorganic Polyphosphate Is a Potential Phosphoryl Group Donor:
For at least one form of the enzyme phosphofructokinase in plants, PPi is the phosphoryl group donor, a role played by ATP in animals and microbes.
In prokaryotes, the enzyme polyphosphate kinase- 1 (PPK-1) catalyzes the reversible reaction.
A second enzyme, polyphosphate kinase-2 (PPK-2), catalyzes the reversible synthesis of GTP (or ATP) from polyphosphate and GDP (or ADP). PPK-2 is believed to act primarily in the direction of GTP and ATP synthesis, and PPK-1 in the direction of polyphosphate synthesis.
PPK-1 and PPK-2 are present in a wide variety of prokaryotes, including many pathogenic bacteria.
SUMMARY:
Direct hydrolysis of ATP is the source of energy in the conformational changes that produce muscle contraction but, in general, it is not ATP hydrolysis but the transfer of a phosphoryl, pyrophosphoryl, or adenylyl group from ATP to a substrate or enzyme molecule that couples the energy of ATP breakdown to endergonic transformations of substrates. Through these group transfer reactions, ATP provides the energy for anabolic reactions, including the synthesis of informational molecules and for the transport of molecules and ions across membranes against concentration gradients and electrical potential gradients.
Thioesters also have high free energies of hydrolysis
Inorganic polyphosphate, present in all cells, may serve as a reservoir of phosphoryl groups with high group transfer potential
GLYCOLYSIS:
A molecule of glucose is degraded in a series of enzyme-catalyzed reactions to yield two molecules of the three-carbon compound pyruvate.
An Overview: Glycolysis Has Two Phases
The breakdown of the six-carbon glucose into two molecules of the three-carbon pyruvate occurs in ten steps.
preparatory phase:
It consists of the first 5 steps. In these reactions, glucose is enzymatically phosphorylated by ATP (first at carbon 6 and later at carbon 1) to yield fructose 1,6- diphosphate which is then split in half to yield 2 moles of the 3-carbon compound, glyceraldehyde 3-phosphate.
The first phase of glycolysis, thus, results in cleavage of the hexose chain. This phase requires an investment of 2ATP moles to activate (or prime) the glucose mole and prepare it for its cleavage into two 3- carbon pieces.
Besides glucose, other hexoses such as D-fructose, D-galactose and D-mannose may also convert into glyceraldehyde 3-phosphate.
B. Phase II \ Payoff Phase:
This phase represents the payoff of glycolysis, in which the energy liberated during conversion of 3 moles of glyceraldehyde 3-phosphate to 2 moles of pyruvate is converted by the coupled phosphorylation of 4 moles of ADP to ATP.
Although 4 moles of ATP are formed in phase II, the net overall yield is only 2 moles of ATP per mole of glucose oxidized, since 2 moles of ATP are invested in phase I. The phase II is, thus, energy conserving.
Importance of Phosphorylated Intermediates Each of the nine glycolytic intermediates between glucose and pyruvate is phosphorylated. The phosphoryl groups appear to have three functions.
1. Because the plasma membrane generally lacks transporters for phosphorylated sugars, the phosphorylated glycolytic intermediates cannot leave the cell. After the initial phosphorylation, no further energy is necessary to retain phosphorylated intermediates in the cell, despite the large difference in their intracellular and extracellular concentrations.
2. Phosphoryl groups are essential components in the enzymatic conservation of metabolic energy. Energy released in the breakage of phosphoanhydride bonds (such as those in ATP) is partially conserved in the formation of phosphate esters such as glucose 6-phosphate. High-energy phosphate compounds formed in glycolysis (1, 3-bisphosphoglycerate and phosphoenolpyruvate) donate phosphoryl groups to ADP to form ATP.
3. Binding energy resulting from the binding of phosphate groups to the active sites of enzymes lowers the activation energy and increases the specificity of the enzymatic reactions. The phosphate groups of ADP, ATP, and the glycolytic intermediates form complexes with Mg2+, and the substrate binding sites of many glycolytic enzymes are specific for these Mg2+ complexes. Most glycolytic enzymes require Mg2+ for activity
1. Phosphorylation of Glucose
Reaction, which is irreversible under intracellular conditions, is catalyzed by Hexokinase.
Recall that Kinases are enzymes that catalyze the transfer of the terminal phosphoryl group from ATP to an acceptor nucleophile.
Kinases are a subclass of Transferases Hexokinase, like many other kinases, requires Mg2+ for its activity, because the true substrate of the enzyme is not ATP4_ but the MgATP2_ complex.
Hexokinase is present in all cells of all organisms.
Hepatocytes also contain a form of hexokinase called hexokinase IV or glucokinase, which differs from other forms of hexokinase in kinetic and regulatory properties.
Glucokinase (often designated hexokinase IV) is a monomeric inducible enzyme (MW = 48,000) and is found almost exclusively in the liver. Glucokinase differs from mammalian hexokinase in 3 respects:
(a) It is specific for glucose and does not act on other hexoses.
(b) It is not inhibited by glucose 6-phosphate.
(c) It has a low affinity (i.e., a much higher Km value of about 10 mM) for glucose than hexokinase.
The function of glucokinase is to remove glucose from the blood following a meal and to trap it in the liver cells, thereby allowing storage of glucose as glycogen or, after further metabolism, as fatty acids
Two enzymes that catalyze the same reaction but are encoded in different genes are called isozymes.
2. Conversion of Glucose 6-Phosphate to Fructose 6-Phosphate
The enzyme phosphohexose isomerase (phosphoglucose isomerase) catalyzes the reversible isomerization of glucose 6-phosphate, an aldose, to fructose 6-phosphate, a ketose.
3. Phosphorylation of Fructose 6-Phosphate to Fructose 1,6- Bisphosphate
In the second of the two priming reactions of glycolysis, phosphofructokinase-1 (PFK-1) catalyzes the transfer of a phosphoryl group from ATP to fructose 6-phosphate to yield fructose 1,6-bisphosphate.
This enzyme is called PFK-1 to distinguish it from a second enzyme (PFK-2) that catalyzes the formation of fructose 2,6-bisphosphate from fructose 6-phosphate in a separate pathway.
Some bacteria and protists and perhaps all plants have a phosphofructokinase that uses pyrophosphate (PPi), not ATP, as the phosphoryl group donor in the synthesis of fructose 1,6-bisphosphate.
Phosphofructokinase-1 is a regulatory enzyme
The activity of PFK-1 is increased whenever the cell’s ATP supply is depleted or when the ATP breakdown products, ADP and AMP (particularly the latter), are in excess.
The enzyme is inhibited whenever the cell has ample ATP and is well supplied by other fuels such as fatty acids.
In some organisms, fructose 2, 6-bisphosphate (not to be confused with the PFK-1 reaction product, fructose 1, 6- bisphosphate) is a potent allosteric activator of PFK-1.
4. Cleavage of Fructose 1,6-Bisphosphate
The enzyme fructose 1, 6-bisphosphate aldolase, often called simply aldolase, catalyzes a reversible aldol condensation.
Fructose 1,6-bisphosphate is cleaved to yield two different triose phosphates, glyceraldehydes 3-phosphate, an aldose, and dihydroxyacetone phosphate, a ketose:
There are two classes of aldolases.
Class I aldolases, found in animals and plants.
Class II enzymes, in fungi and bacteria, do not form the Schiff base intermediate. Instead, a zinc ion at the active site is coordinated with the carbonyl oxygen at C-2; the Zn2+ polarizes the carbonyl group and stabilizes the enolate intermediate created in the C-C bond cleavage step.
5. Interconversion of the Triose Phosphates
Only one of the two triose phosphates formed by aldolase, glyceraldehydes 3-phosphate, can be directly degraded in the subsequent steps of glycolysis.
The other product, dihydroxyacetone phosphate, is rapidly and reversibly converted to glyceraldehyde 3-phosphate by the fifth enzyme of the sequence, triose phosphate isomerase:
2.The Payoff Phase of Glycolysis Yields ATP and NADH
The conversion of two molecules of glyceraldehyde 3-phosphate to two molecules of pyruvate is accompanied by the formation of four molecules of ATP from ADP.
However, the net yield of ATP per molecule of glucose degraded is only two, because two ATP were invested in the preparatory phase of glycolysis to phosphorylate the two ends of the hexose molecule.
6. Oxidation of Glyceraldehyde 3-Phosphate to 1,3-Bisphosphoglycerate:
The first step in the payoff phase is the oxidation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate, catalyzed by glyceraldehyde 3- phosphate dehydrogenase:
The aldehyde group of glyceraldehyde 3-phosphate is oxidized, not to a free carboxyl group but to a carboxylic acid anhydride with phosphoric acid.
This type of anhydride, called an acyl phosphate, has a very high standard free energy of hydrolysis .
The reduction of NAD+proceeds by the enzymatic transfer of a hydride ion (:H+) from the aldehyde group of glyceraldehyde 3-phosphate to the nicotinamide ring of NAD+, yielding the reduced coenzyme NADH.
The other hydrogen atom of the substrate molecule is released to the solution as H+.
The aldehyde group of glyceraldehyde 3-phosphate reacts with the -SH group of an essential Cys residue in the active site, in a reaction analogous to the formation of a hemiacetal, in this case producing a thiohemiacetal.
Reaction of the essential Cys residue with a heavy metal such as Hg2+ irreversibly inhibits the enzyme.
Because cells maintain only limited amounts of NAD+, glycolysis would soon come to a halt if the NADH formed in this step of glycolysis were not continuously reoxidized.
7.Phosphoryl Transfer from 1,3-Bisphosphoglycerate to ADP
The enzyme phosphoglycerate kinase transfers the high-energy phosphoryl group from the carboxyl group of 1,3-bisphosphoglycerate to ADP, forming ATP and 3- phosphoglycerate:
Steps 6 and 7 of glycolysis together constitute an Energy-coupling process in which 1,3-bisphosphoglycerate is the common intermediate; it is formed in the first reaction (which would be endergonic in isolation), and its acyl phosphate group is transferred to ADP in the second reaction (which is strongly exergonic). The
sum of these two reactions is-----
Thus the overall reaction is exergonic.
For step 6-----
Notice that [H+] is not included in Q. In biochemical calculations, [H+] is assumed to be a constant (10-7 M), and this constant is included in the definition of ∆G◦.
The formation of ATP by phosphoryl group transfer from a substrate such as 1,3-bisphosphoglycerate is referred to as a substrate-level phosphorylation, to distinguish this mechanism from respiration-linked phosphorylation.
Substrate-level phosphorylations involve soluble enzymes and chemical intermediates (1,3-bisphosphoglycerate in this case).
Respiration-linked phosphorylations, on the other hand, involve membrane-bound enzymes and transmembrane gradients of protons.
8. Conversion of 3-Phosphoglycerate to 2-Phosphoglycerate
The enzyme phosphoglycerate mutase catalyzes a reversible shift of the phosphoryl group between C-2 and C-3 of glycerate; Mg2+ is essential for this reaction:
The reaction occurs in two steps. A phosphoryl group initially attached to a His residue of the mutase is transferred to the hydroxyl group at C-2 of 3- phosphoglycerate, forming 2,3-bisphosphoglycerate (2,3-BPG). The phosphoryl group at C-3 of 2,3-BPG is then transferred to the same His residue, producing 2- phosphoglycerate and regenerating the phosphorylated enzyme.
Although in most cells 2,3-BPG is present in only trace amounts, it is a major component (~5 mM) of erythrocytes, where it regulates the affinity of hemoglobin for oxygen.
9. Dehydration of 2-Phosphoglycerate to Phosphoenolpyruvate:
In the second glycolytic reaction that generates a compound with high phosphoryl group transfer potential, enolase promotes reversible removal of a molecule of water from 2-phosphoglycerate to yield phosphoenolpyruvate (PEP):
Although 2-phosphoglycerate and phosphoenolpyruvate contain nearly the same total amount of energy, the loss of the water molecule from 2-phosphoglycerate causes a redistribution of energy within the molecule, greatly increasing the standard free energy of hydrolysis of the phosphoryl group.
10. Transfer of the Phosphoryl Group from Phosphoenolpyruvate to ADP
The last step in glycolysis is the transfer of the phosphoryl group from phosphoenolpyruvate to ADP, catalyzed by pyruvate kinase, which requires K+ and either Mg2+ or Mn2+:
In this substrate-level phosphorylation, the product pyruvate first appears in its enol form, then tautomerizes rapidly and nonenzymatically to its keto form, which predominates at pH 7:
The overall reaction has a large, negative standard free energy change, due in large part to the spontaneous conversion of the enol form of pyruvate to the keto form.
The Overall Balance Sheet Shows a Net Gain of ATP
The two molecules of NADH formed by glycolysis in the cytosol are, under aerobic conditions, reoxidized to NAD+ by transfer of their electrons to the electrontransfer chain, which in eukaryotic cells is located in the mitochondria. The electron-transfer chain passes these electrons to their ultimate destination, O2:
Glycolysis Is under Tight Regulation
The ATP yield from glycolysis under anaerobic conditions (2 ATP per molecule of glucose) is much smaller than that from the complete oxidation of glucose to CO2 under aerobic conditions (30 or 32 ATP per glucose; see Table 19–5). About 15 times as much glucose must therefore be consumed anaerobically as aerobically to yield the same amount of ATP.
Cancerous Tissue Has Deranged Glucose Catabolism
Glucose uptake and glycolysis proceed about ten times faster in most solid tumors than in noncancerous tissues.
Tumor cells commonly experience hypoxia (limited oxygen supply), because they initially lack an extensive capillary network to supply the tumor with oxygen. As a result, cancer cells more than 100 to 200 µm from the nearest capillaries depend on anaerobic glycolysis for much of their ATP production.
They take up more glucose than normal cells, converting it to pyruvate and then to lactate as they recycle NADH.
SUMMARY
■ All ten glycolytic enzymes are in the cytosol, and all ten intermediates are phosphorylated compounds of three or six carbons.
■ In the preparatory phase of glycolysis, ATP is invested to convert glucose to fructose 1, 6-bisphosphate. The bond between C-3 and C-4 is then broken to yield two molecules of triose phosphate.
■ In the payoff phase, each of the two molecules of glyceraldehyde 3-phosphate derived from glucose undergoes oxidation at C-1; the energy of this oxidation reaction is conserved in the formation of one NADH and two ATP per triose phosphate oxidized.
■ Glycolysis is tightly regulated in coordination with other energy-yielding pathways to assure a steady supply of ATP. Hexokinase, PFK-1, and pyruvate kinase are all subject to allosteric regulation that controls the flow of carbon through the pathway and maintains constant levels of metabolic intermediates.
THE CITRIC ACID CYCLE:
Cellular respiration occurs in three major stages.
In the first, organic fuel molecules—glucose, fatty acids, and some amino acids—are oxidized to yield two-carbon fragments in the form of the acetyl group of acetyl-coenzyme A (acetyl-CoA).
In the second stage, the acetyl groups are fed into the citric acid cycle, which enzymatically oxidizes them to CO2; the energy released is conserved in the reduced electron carriers NADH and FADH2.
In the third stage of respiration, these reduced coenzymes are themselves oxidized, giving up protons (H+) and electrons. The electrons are transferred to O2—the final electron acceptor—via a chain of electron-carrying molecules known as the respiratory chain. In the course of electron transfer, the large amount of energy released is conserved in the form of ATP, by a process called oxidative phosphorylation
Production of Acetyl-CoA (Activated Acetate):
Pyruvate, derived from glucose and other sugars by glycolysis, is oxidized to acetyl-CoA and CO2 by the pyruvate dehydrogenase (PDH) complex
(PDH) is a cluster of enzymes—multiple copies of each of three enzymes—located in the mitochondria of eukaryotic cells and in the cytosol of prokaryotes.
The PDH complex is the prototype for two other important enzyme complexes: α-ketoglutarate dehydrogenase, of the citric acid cycle, and the branched-chain α-keto acid dehydrogenase, of the oxidative pathways of several amino acids.
PDH need five cofactors, four derived from vitamins, participate in the reaction mechanism. (Thiamine pyrophosphate (TPP), FAD, coenzyme A, NAD and lipoate. Four different vitamins thiamine (in TPP), riboflavin (in FAD), niacin (in NAD), and pantothenate (in CoA).
Because of its capacity to undergo oxidation-reduction reactions, lipoate can serve both as an electron hydrogen carrier and as an acyl carrier.
Pyruvate Is Oxidized to Acetyl-CoA and CO2
The overall reaction catalyzed by the pyruvate dehydrogenase complex is an oxidative decarboxylation, an irreversible oxidation process in which the carboxyl group is removed from pyruvate as a molecule of CO2 and the two remaining carbons become the acetyl group of acetyl-CoA.
The Pyruvate Dehydrogenase Complex Consists of Three Distinct Enzymes:
The PDH complex is composed of multiple copies of three enzymes: pyruvate dehydrogenase, E1 (with its bound cofactor TPP); dihydrolipoyl transacetylase, E2 (with its covalently bound lipoyl group); and dihydrolipoyl dehydrogenase, E3 (with its cofactors FAD and NAD).
E1 catalyzes first the decarboxylation of pyruvate, producing hydroxyethyl-TPP, and then the oxidation of the hydroxyethyl group to an acetyl group. The electrons from this oxidation reduce the disulfide of lipoate bound to E2, and the acetyl group is transferred into thioester linkage with one -SH group of reduced lipoate.
E2 catalyzes the transfer of the acetyl group to coenzyme A, forming acetyl-CoA.
E3 catalyzes the regeneration of the disulfide (oxidized) form of lipoate; electrons pass first to FAD, then to NAD+
The long lipoyllysine arm swings from the active site of E1 to E2 to E3, tethering the intermediates to the enzyme complex to allow substrate channeling
Reactions of the Citric Acid Cycle:
The Citric Acid Cycle Has Eight Steps
1,Formation of Citrate
The first reaction of the cycle is the condensation of acetyl-CoA with oxaloacetate to form citrate, catalyzed by citrate synthase:
The large, negative standard free-energy change of the citrate synthase reaction is essential to the operation of the cycle because, as noted earlier, the concentration of oxaloacetate is normally very low.
The CoA liberated in this reaction is recycled to participate in the oxidative decarboxylation of another molecule of pyruvate by the PDH complex.
The reaction catalyzed by citrate synthase is essentially a Claisen condensation, involving a thioester (acetyl-CoA) and a ketone (oxaloacetate).
2.Formation of Isocitrate via cis-Aconitate:
The enzyme aconitase (more formally, aconitate hydratase) catalyzes the reversible transformation of citrate to isocitrate, through the intermediary formation of the tricarboxylic acid cis-aconitate, which normally does not dissociate from the active site.
Aconitase can promote the reversible addition of H2O to the double bond of enzyme-bound cis-aconitate in two different ways, one leading to citrate and the other to isocitrate:
Aconitase contains an ironsulfur center , which acts both in the binding of the substrate at the active site and in the catalytic addition or removal of H2O.
3.Oxidation of Isocitrate to _-Ketoglutarate and CO2
isocitrate dehydrogenase catalyzes oxidative decarboxylation of isocitrate to form α-ketoglutarate.
Mn2+in the active site interacts with the carbonyl group of the intermediate oxalosuccinate, which is formed transiently .
Mn2+ also stabilizes the enol formed transiently by decarboxylation.
There are two different forms of isocitrate dehydrogenase in all cells, one requiring NAD+as electron acceptor and the other requiring NADP+.
In eukaryotic cells, the NAD-dependent enzyme occurs in the mitochondrial matrix and serves in the citric acid cycle.
4.Oxidation of α-Ketoglutarate to Succinyl-CoA and CO2
The next step is another oxidative decarboxylation, in which α-ketoglutarate is converted to succinyl-CoA and CO2 by the action of the α-ketoglutarate dehydrogenase complex.
NAD+ serves as electron acceptor and CoA as the carrier of the succinyl group.
The energy of oxidation of α-ketoglutarate is conserved in the formation of the thioester bond of succinyl-CoA:
This reaction is virtually identical to the pyruvate dehydrogenase reaction discussed above, and the α-ketoglutarate dehydrogenase complex closely resembles the PDH complex in both structure and function.
5. Conversion of Succinyl-CoA to Succinate
Succinyl-CoA, like acetyl-CoA, has a thioester bond with a strongly negative standard free energy of hydrolysis (∆G◦= 36 kJ/mol).
In the next step of the citric acid cycle, energy released in the breakage of this bond is used to drive the synthesis of a phosphoanhydride bond in either GTP or ATP
Succinate is formed in the process
The enzyme that catalyzes this reversible reaction is called succinyl-CoA synthetase or succinic thiokinase; both names indicate the participation of a nucleoside triphosphate in the reaction.
Catalyse by nucleoside diphosphate kinase
There is no change in free energy for the nucleoside diphosphate kinase reaction; ATP and GTP are energetically equivalent.
6. Oxidation of Succinate to Fumarate
The succinate formed from succinyl-CoA is oxidized to fumarate by the flavoprotein succinate dehydrogenase:
In eukaryotes, succinate dehydrogenase is tightly bound to the inner mitochondrial membrane; in prokaryotes, to the plasma membrane.
Succinate dehydrogenase differs from other enzymes in the citric acid cycle in that it is an integral part of the inner mitochondrial membrane
Malonate, an analog of succinate not normally present in cells, is a strong competitive inhibitor of succinate dehydrogenase and its addition to mitochondria blocks the activity of the citric acid cycle.
7. Hydration of Fumarate to Malate
The reversible hydration of fumarate to L-malate is catalyzed by fumarase( formally, fumarate hydratase). The transition state in this reaction is a carbanion:
This enzyme is highly stereospecific; it catalyzes hydration of the trans double bond of fumarate but not the cis double bond of maleate (the cis isomer of fumarate).
8. Oxidation of Malate to Oxaloacetate
In the last reaction of the citric acid cycle, NAD-linked L-malate dehydrogenase catalyzes the oxidation of L-malate to oxaloacetate.
.
The citric acid cycle and the connected reactions consume 4 moles of water and release 5 moles.
In the absence of oxygen, the citric acid cycle is inactive, and less ATP is generated per molecule of glucose.
Q---Although oxygen does not participate directly in the citric acid cycle, the cycle operates only when O2 is present. Why?
A----Oxygen is needed to recycle NAD+ from the NADH produced by the oxidative reactions of the citric acid cycle. Reoxidation of NADH occurs during mitochondrial oxidative Phosphorylation
Acetyl-CoA enters the citric acid cycle (in the mitochondria of eukaryotes, the cytosol of prokaryotes).
STOICHIOMETRY OF THE CITRIC ACID CYCLE
The ∆Gºfor this overall reaction is –14.3 kcal/mol.
Four pairs of hydrogen atoms are removed from the four cycle intermediates by enzymatic dehydrogenation (Steps 3, 4, 6 and 8). Three pairs of hydrogen are used to reduce 3 moles of NAD+ to NADH and one pair to reduce the FAD of succinate dehydrogenase to FADH2.
It is interesting to note that molecular oxygen deos not participate directly in the citric acid cycle. However, the cycle operates only under aerobic conditions because NAD+ and FAD can be regenerated from their reduced forms (NADH and FADH2) in the mitochondrion only by electron transfer to molecular oxygen. Thus, whereas glycolysis has an aerobic and an anaerobic nature, the citric acid cycle is strictly aerobic in nature.
Citric Acid Cycle Components Are Important Biosynthetic Intermediates
In aerobic organisms, the citric acid cycle is an amphibolic pathway, one that serves in both catabolic and anabolic processes.
Besides its role in the oxidative catabolism of carbohydrates, fatty acids, and amino acids, the cycle provides precursors for many biosynthetic pathways. α-Ketoglutarate and oxaloacetate can, for example, serve as precursors of the amino acids aspartate and glutamate by simple transamination.
Oxaloacetate is converted to glucose in gluconeogenesis .
Succinyl- CoA is a central intermediate in the synthesis of the porphyrin ring of heme groups, which serve as oxygen carriers (in hemoglobin and myoglobin) and electron carriers (in cytochromes).
Anaplerotic Roles;
Anaplerosis is defined as any reaction that can restore the concentration of a crucial but depleted intermediate.
The pyruvate carboxylase reaction requires the vitamin biotin , which is the prosthetic group of the enzyme. Biotin plays a key role in many carboxylation reactions. It is a specialized carrier of one-carbon groups in their most oxidized form: CO2.
Regulation of the Citric Acid Cycle
FIGURE 16–18 Regulation of metabolite flow from the PDH complex through the citric
acid cycle.
The PDH complex is allosterically inhibited when [ATP]/[ADP], [NADH]/[NAD+], and [acetyl-CoA]/[CoA] ratios are high, indicating an energy-sufficient metabolic state.
When these ratios decrease, allosteric activation of pyruvate oxidation results. The rate of flow through the citric acid cycle can be limited by the availability of the citrate synthase substrates, oxaloacetate and acetyl-CoA, or of NAD+, which is depleted by its conversion to NADH, slowing the three NAD+dependent oxidation steps.
Feedback inhibition by succinyl-CoA, citrate, and ATP also slows the cycle by inhibiting early steps. In muscle tissue, Ca2+ signals contraction and, as shown here, stimulates energy-yielding metabolism to replace the ATP consumed by contraction.
Coenzyme Levels
As a general rule, catabolic energy-yielding processes generally require NAD+, while anabolic energy-requiring ones almost invariably require NADP+. These two coenzymes interact with one another according to the following equation.
When NAD+/NADH ratio is high, the rate of citric acid cycle becomes rapid. However, the activity of this cycle is retarded when NAD+/NADH ratio is low because of (a) insufficient NAD+ concentration for otherwise normal enzymatic function, and (b) reoxidation of NADH coupled to ATP formation.
The Glyoxylate Cycle
The glyoxylate cycle is active in the germinating seeds of some plants and in certain microorganisms that can live on acetate as the sole carbon source.
In plants, the pathway takes place in glyoxysomes in seedlings. It involves several citric acid cycle enzymes and two additional enzymes: isocitrate lyase and malate synthase.
The partitioning of isocitrate between the citric acid cycle and the glyoxylate cycle is controlled at the level of isocitrate dehydrogenase, which is regulated by reversible phosphorylation.
Vertebrates lack the glyoxylate cycle and cannot synthesize glucose from acetate or the fatty acids that give rise to acetyl-CoA.
OXIDATIVE PHOSPHORYLATION:
Oxidative phosphorylation involves the reduction of O2 to H2O with electrons donated by NADH and FADH2; it occurs equally well in light or darkness.
Photophosphorylation involves the oxidation of H2O to O2, with NADP+ as ultimate electron acceptor; it is absolutely dependent on the energy of light.
In eukaryotes, oxidative phosphorylation occurs in mitochondria, Photophosphorylation in chloroplasts.
Electron-Transfer Reactions in Mitochondria:
The outer mitochondrial membrane is readily permeable to small molecules (Mr <5,000) and ions, which move freely through transmembrane channels formed by a family of integral membrane proteins called porins.
The inner membrane is impermeable to most small molecules and ions, including protons (H+); the only species that cross this membrane do so through specific transporters.
The inner membrane bears the components of the respiratory chain and the ATP synthase.
The mitochondrial matrix, enclosed by the inner membrane, contains the pyruvate dehydrogenase complex and the enzymes of the citric acid cycle, the fatty acid ,B-oxidation pathway, and the pathways of amino acid oxidation—all the pathways of fuel oxidation except glycolysis, which takes place in the cytosol.
Electrons Are Funneled to Universal Electron Acceptors:
universal electron acceptors—nicotinamide nucleotides (NAD+or NADP+) or flavin nucleotides (FMN or FAD).
Nicotinamide nucleotide–linked dehydrogenases catalyze reversible reactions of the following general types-
NAD+ linked dehydrogenases remove two hydrogen atoms from their substrates. One of these is transferred as a hydride ion (: H-) to NAD+; the other is released as H+ in the medium.
NADH and NADPH are water-soluble electron carriers that associate reversibly with dehydrogenases.
NADH carries electrons from catabolic reactions. NADPH generally supplies electrons to anabolic reactions.
Cells maintain separate pools of NADPH and NADH, with different redox potentials.
This is accomplished by holding the ratios of [reduced form]/[oxidized form] relatively high for NADPH and relatively low for NADH. Neither NADH nor NADPH can cross the inner mitochondrial membrane, but the electrons they carry can be shuttled across indirectly,
Flavoproteins contain a very tightly, sometimes covalently, bound flavin nucleotide, either FMN or FAD.
Electron transfer occurs because the flavoproteins have a higher reduction potential than the compound oxidized. The standard reduction potential of a flavin nucleotide, unlike that of NAD or NADP, depends on the protein with which it is associated.
Electrons Pass through a Series of Membrane-Bound Carriers:
Three types of electron transfers occur in oxidative phosphorylation:
(1) Direct transfer of electrons, as in the reduction of Fe3+ to Fe2+;
(2) transfer as a hydrogen atom (H+ + e-).
(3) Transfer as a hydride ion (:H-), which bears two electrons.
The term reducing equivalent is used to designate a single electron equivalent transferred in an oxidation-reduction reaction.
In addition to NAD and flavoproteins, three other types of electron-carrying molecules function in the respiratory chain: a hydrophobic quinine (ubiquinone) and two different types of iron-containing proteins (cytochromes and iron sulfur proteins).
Ubiquinone (also called coenzyme Q, or simply Q) is a lipid-soluble benzoquinone with a long isoprenoid side chain.
The closely related compounds plastoquinone (of plant chloroplasts) and menaquinone (of bacteria) play roles analogous to that of ubiquinone, carrying electrons in membrane-associated electron-transfer chains.
Ubiquinone can accept one electron to become the semiquinone radical (◦QH) or two electrons to form ubiquinol (QH2) and, like flavoprotein carriers, it can act at the junction between a two-electron donor and a one-electron acceptor.it carries both electrons and protons, it plays a central role in coupling electron flow to proton movement.
The cytochromes are proteins with characteristic strong absorption of visible light, due to their iron containing heme prosthetic groups.
Mitochondria contain three classes of cytochromes, designated a, b, and c, which are distinguished by differences in their light-absorption spectra.
The longest wavelength band is near 600 nm in type a cytochromes near 560 nm in type b, and near 550 nm in type c.
The heme cofactors of a and b cytochromes are tightly, but not covalently, bound to their associated proteins; the hemes of c-type cytochromes are covalently attached through Cys residues.
In iron-sulfur proteins the iron is present not in heme but in association with inorganic sulfur atoms or with the sulfur atoms of Cys residues in the protein, or both.
These iron-sulfur (Fe-S) centers range from simple structures with a single Fe atom coordinated to four Cys -SH groups to more complex Fe-S centers with two or four Fe atoms.
Rieske iron-sulfur proteins (named after their discoverer, John S. Rieske) are a variation on this theme, in which one Fe atom is coordinated to two His residues rather than two Cys residues.
All iron-sulfur proteins participate in one-electron transfers in which one iron atom of the iron-sulfur cluster is oxidized or reduced.
Effects of inhibitors of electron transfer:
Complex I: NADH to Ubiquinone :
Complex I, also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase, is a large enzyme composed of 42 different polypeptide chains, including an FMN-containing flavoprotein and at least six iron sulfur centers.
Complex I catalyzes two simultaneous and obligately coupled processes:
(1) The exergonic transfer to ubiquinone of a hydride ion from NADH and a proton from the matrix, expressed by
(2) The endergonic transfer of four protons from the matrix to the intermembrane space.
Complex I is therefore a proton pump driven by the energy of electron transfer, and the reaction it catalyzes is vectorial: it moves protons in a specific direction from one location (the matrix, which becomes negatively charged with the departure of protons) to another (the intermembrane space, which becomes positively charged).
NADH:ubiquinone oxidoreductase (Complex I). Complex I catalyzes the transfer of a hydride ion from NADH to FMN, from which two electrons pass through a series of Fe-S centers to the ironsulfur protein N-2 in the matrix arm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2, which diffuses into the lipid bilayer. This electron transfer also drives the expulsion from the matrix of four protons per pair of electrons.
Amytal (a barbiturate drug), rotenone (a plant product commonly used as an insecticide), and piericidin A (an antibiotic) inhibit electron flow from the Fe-S centers of Complex I to ubiquinone and therefore block the overall process of oxidative phosphorylation.
Complex II: Succinate to Ubiquinone:
Complex II in Chapter 16 as succinate dehydrogenase, the only membrane-bound enzyme in the citric acid cycle.
Although smaller and simpler than Complex I, it contains five prosthetic groups of two types and four different protein subunits.
The path of electron transfer from the succinate-binding site to FAD, then through the Fe-S centers to the Q-binding site, is more than 40 Å long, but none of the individual electron-transfer distances exceeds about 11 Å—a reasonable distance for rapid electron transfer.
Complex III: Ubiquinone to Cytochrome c:
Complex III, also called cytochromes bc1 complex or ubiquinone:cytochrome c oxidoreductase, couples the transfer of electrons from ubiquinol (QH2) to cytochrome c with the vectorial transport of protons from the matrix to the intermembrane space.
The net equation for the redox reactions of this Q cycle
The Q cycle accommodates the switch between the two electron carrier ubiquinone and the one-electron carriers— cytochromes b562, b566, c1, and c—and explains the measured stoichiometry of four protons translocated per pair of electrons passing through the Complex III to cytochrome c.
QH2 is oxidized to Q and two molecules of cytochrome c are reduced.
cytochrome c moves to Complex IV to donate the electron to a binuclear copper center.
Complex IV: Cytochrome c to O2:
Complex IV, also called cytochromes oxidase, carries electrons from cytochrome c to molecular oxygen, reducing it to H2O.
Path of electrons through Complex IV.
The three proteins critical to electron flow are subunits I, II, and III. The larger green structure includes the other ten proteins in the complex.
Electron transfer through Complex IV begins when two molecules of reduced cytochromes c (top) each donate an electron to the binuclear center CuA. From here electrons pass through heme a to the Fe-Cu center (cytochromes a3 and CuB). Oxygen now binds to heme a3 and is reduced to its peroxy derivative (O2 2-by two electrons from the Fe-Cu center. Delivery of two more electrons from cytochrome c (making four electrons in all) converts the O2 2_ to two molecules of water, with consumption of four “substrate” protons from the matrix. At the same time, four more protons are pumped from the matrix by an as yet unknown mechanism.
Plant Mitochondria Have Alternative Mechanisms for Oxidizing NADH:
In the light, the principal source of mitochondrial NADH is a reaction in which glycine, produced by a process known as photorespiration, is converted to serine.
For reasons, plants must carry out this reaction even when they do not need NADH for ATP production. To regenerate NAD+ from unneeded NADH, plant mitochondria transfer electrons from NADH directly to ubiquinone and from ubiquinone directly to O2, bypassing Complexes III and IV and their proton pumps.
Unlike cytochrome oxidase (Complex IV), the alternative QH2 oxidase is not inhibited by cyanide.
Cyanide-resistant NADH oxidation is therefore the hallmark of this unique plant electron-transfer pathway.
The Energy of Electron Transfer Is Efficiently Conserved in a Proton Gradient::
The transfer of two electrons from NADH through the respiratory chain to molecular oxygen can be written as
This net reaction is highly exergonic. Much of this energy is used to pump protons out of the matrix. For each pair of electrons transferred to O2, four protons are pumped out by Complex I, four by Complex III, and two by Complex IV. The vectorial equation for the process is therefore-----
The electrochemical energy inherent in this difference in proton concentration and separation of charge represents a temporary conservation of much of the energy of electron transfer. The energy stored in such a gradient, termed the proton-motive force, has two components:
(1) The chemical potential energy due to the difference in concentration of a chemical species (H+) in the two regions separated by the membrane.
(2) The electrical potential energy that results from the separation of charge when a proton moves across the membrane without a counter ion.
When protons flow spontaneously down their electrochemical gradient, energy is made available to do work.
In mitochondria, chloroplasts, and aerobic bacteria, the electrochemical energy in the proton gradient drives the synthesis of ATP from ADP and Pi.
ATP SYNTHESIS::
How is a concentration gradient of protons transformed into ATP?
The chemiosmotic model.
FIGURE 19–17 Chemiosmotic model. In this simple representation of the chemiosmotic theory applied to mitochondria, electrons from NADH and other oxidizable substrates pass through a chain of carriers arranged asymmetrically in the inner membrane. Electron flow is accompanied by proton transfer across the membrane, producing both a chemical gradient (∆pH) and an electrical gradient (∆Ψ). The inner mitochondrial membrane is impermeable to protons; protons can reenter the matrix only through proton-specific channels (Fo). The proton-motive force that drives protons back into the matrix provides the energy for ATP synthesis, catalyzed by the F1 complex associated with Fo.
To emphasize this crucial role of the protonmotive force, the equation for ATP synthesis is sometimes written—
When isolated mitochondria are suspended in a buffer containing ADP, Pi, and an oxidizable substrate such as succinate, three easily measured processes occur:
(1) The substrate is oxidized (succinate yields fumarate).
(2) O2 is consumed.
(3) ATP is synthesized.
Oxygen consumption and ATP synthesis depend on the presence of an oxidizable substrate (succinate in this case) as well as ADP and Pi. Because the energy of substrate oxidation drives ATP synthesis in mitochondria.
Inhibition of ATP synthesis blocks electron transfer in intact mitochondria.
This obligatory coupling can be demonstrated in isolated mitochondria by providing O2 and oxidizable substrates, but not ADP . Under these conditions, no ATP synthesis can occur and electron transfer to O2 does not proceed.
Coupling of oxidation and phosphorylation can also be demonstrated using oligomycin or venturicidin, toxic antibiotics that bind to the ATP synthase in mitochondria.
These compounds are potent inhibitors of both ATP synthesis and the transfer of electrons through the chain of carriers to O2 .
Because oligomycin is known to interact not directly with the electron carriers but with ATP synthase, it follows that electron transfer and ATP synthesis are obligately coupled; neither reaction occurs without the other.
FIGURE 19–18 Coupling of electron transfer and ATP synthesis in mitochondria. In experiments to demonstrate coupling, mitochondria are suspended in a buffered medium and an O2 electrode monitors O2 consumption. At intervals, samples are removed and assayed for the presence of ATP. (a) Addition of ADP and Pi alone results in little or no increase in either respiration (O2 consumption; black) or ATP synthesis (red). When succinate is added, respiration begins immediately and ATP is synthesized. Addition of cyanide (CN-), which blocks electron transfer between cytochrome oxidase and O2, inhibits both respiration and ATP synthesis. (b) Mitochondria provided with succinate respire and synthesize ATP only when ADP and Pi are added. Subsequent addition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks both ATP synthesis and respiration. Dinitrophenol (DNP) carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) is an uncoupler, allowing respiration to continue without ATP synthesis.
Mitochondria manipulated so as to impose a difference of proton concentration and a separation of charge across the inner membrane synthesize ATP in the absence of an oxidizable substrate; the proton-motive force alone suffices to drive ATP synthesis.
ATP Synthase Has Two Functional Domains, Fo and F1::
Mitochondrial ATP synthase is an F-type ATPase similar in structure and mechanism to the ATP synthases of chloroplasts and eubacteria.
This large enzyme complex of the inner mitochondrial membrane catalyzes the formation of ATP from ADP and Pi, accompanied by the flow of protons from the P to the N side of the membrane.
ATP synthase, also called Complex V, has two distinct components: F1, a peripheral membrane protein, and Fo (o denoting oligomycin-sensitive), which is integral to the membrane.
When F1 is gently extracted, the “stripped” vesicles still contain intact respiratory chains and the Fo portion of ATP synthase. The vesicles can catalyze electron transfer from NADH to O2 but cannot produce a proton gradient: Fo has a proton pore through which protons leak as fast as they are pumped by electron transfer, and without a proton gradient the F1-depleted vesicles cannot make ATP.
Isolated F1 catalyzes ATP hydrolysis (the reversal of synthesis) and was therefore originally called F1ATPase. When purified F1 is added back to the depleted vesicles, it reassociates with Fo, plugging its proton pore and restoring the membrane’s capacity to couple electron transfer and ATP synthesis.
ATP IS STABILIZED RELATIVE TO ADP ON THE SURFACE OF F1
Isotope exchange experiments with purified F1 reveal a remarkable fact about the enzyme’s catalytic mechanism: on the enzyme surface, the reaction is readily reversible—the free-energy change for ATP synthesis is close to zero.
ATP synthase stabilizes ATP relative to ADP +Pi by binding ATP more tightly, releasing enough energy to counterbalance the cost of making ATP. Careful measurements of the binding constants show that FoF1 binds ATP with very high affinity (Kd 10-12 M) and ADP with much lower affinity (Kd 10-5 M). The difference in Kd corresponds to a difference of about 40 kJ/mol in binding energy, and this binding energy drives the equilibrium toward formation of the product ATP.
The Proton Gradient Drives the Release of ATP from the Enzyme Surface
It is the proton gradient that causes the enzyme to release the ATP formed on its surface.
Chemiosmotic Coupling Allows Nonintegral Stoichiometries of O2 Consumption and ATP Synthesis
Before the general acceptance of the chemiosmotic model for oxidative phosphorylation, the assumption was that the overall reaction equation would take the following form
with the value of x—sometimes called the P/O ratio or the P/2e_ ratio—always an integer. ATP synthesis is readily measurable, as is the decrease in O2.
Most experiments have yielded P/O (ATP to 1/2O2) ratios of between 2 and 3 when NADH was the electron donor and between 1 and 2 when succinate was the donor.
The most widely accepted experimental value for number of protons required to drive the synthesis of an ATP molecule is 4, of which 1 is used in transporting Pi, ATP, and ADP across the mitochondrial membrane . If 10 protons are pumped out per NADH and 4 must flow in to produce 1 ATP, the proton-based P/O ratio is 2.5 for NADH as the electron donor and 1.5 (6/4) for succinate.
The Proton-Motive Force Energizes Active Transport
The adenine nucleotide translocase, integral to the inner membrane, binds ADP3_ in the intermembrane space and transports it into the matrix in exchange for an ATP4_ molecule simultaneously transported outward. Because this antiporter moves four negative charges out for every three moved in,
A second membrane transport system essential to oxidative phosphorylation is the phosphate translocase, which promotes symport of one H2PO4 _ and one H+ into the matrix.
A complex of the ATP synthase and both translocases, the ATP synthasome,
SHUTTLE SYSTEMS INDIRECTLY CONVEY CYTOSOLIC NADH INTO MITOCHONDRIA FOR OXIDATION
The NADH dehydrogenase of the inner mitochondrial membrane of animal cells can accept electrons only from NADH in the matrix. Given that the inner membrane is not permeable to NADH, how can the NADH generated by glycolysis in the cytosol be reoxidized to NAD+by O2 via the respiratory chain?
Special shuttle systems carry reducing equivalents from cytosolic NADH into mitochondria by an indirect route. The most active NADH shuttle, which functions in liver, kidney, and heart mitochondria, is the malate-aspartate shuttle.
FIGURE 19–27 Malate-aspartate shuttle. This shuttle for transporting reducing equivalents from cytosolic NADH into the mitochondrial matrix is used in liver, kidney, and heart. 1 NADH in the cytosol (intermembrane space) passes two reducing equivalents to oxaloacetate, producing malate. 2 Malate crosses the inner membrane via the malateα ketoglutarate transporter. 3 In the matrix, malate passes two reducing equivalents to NAD+, and the resulting NADH is oxidized by the respiratory chain. The oxaloacetate formed from malate cannot pass directly into the cytosol. 4 It is first transaminated to aspartate, which 5 can leave via the glutamate-aspartate transporter. 6 Oxaloacetate is regenerated in the cytosol, completing the cycle.
Skeletal muscle and brain use a different NADH shuttle, the glycerol 3-phosphate shuttle . It differs from the malate-aspartate shuttle in that it delivers the reducing equivalents from NADH to ubiquinone and thus into Complex III, not Complex I (Fig. 19–8), providing only enough energy to synthesize 1.5 ATP molecules per pair of electrons.
FIGURE 19–28 Glycerol 3-phosphate shuttle. This alternative means of moving reducing equivalents from the cytosol to the mitochondrial matrix operates in skeletal muscle and the brain. In the cytosol, dihydroxyacetone phosphate accepts two reducing equivalents from NADH in a reaction catalyzed by cytosolic glycerol 3-phosphate dehydrogenase. An isozymes of glycerol 3-phosphate dehydrogenase bound to the outer face of the inner membrane then transfers two reducing equivalents from glycerol 3-phosphate in the intermembrane space to ubiquinone. Note that this shuttle does not involve membrane transport systems.
SUMMARY
The flow of electrons through Complexes I, III, and IV results in pumping of protons across the inner mitochondrial membrane, making the matrix alkaline relative to the intermembrane space. This proton gradient provides the energy (in the form of the proton-motive force) for ATP synthesis from ADP and Pi by ATP synthase (FoF1 complex) in the inner membrane.
ATP synthase carries out “rotational catalysis,” in which the flow of protons through Fo causes each of three nucleotide-binding sites in F1 to cycle from (ADP +Pi)–bound to ATP-bound to empty conformations.
■ ATP formation on the enzyme requires little energy; the role of the proton-motive force is to push ATP from its binding site on the synthase.
■ The ratio of ATP synthesized per 1/2O2 reduced to H2O (the P/O ratio) is about 2.5 when electrons enter the respiratory chain at Complex I, and 1.5 when electrons enter at CoQ.
■ Energy conserved in a proton gradient can drive solute transport uphill across a membrane.
■ The inner mitochondrial membrane is impermeable to NADH and NAD_, but NADH equivalents are moved from the cytosol to the matrix by either of two shuttles. NADH equivalents moved in by the malate-aspartate shuttle enter the respiratory chain at Complex I and yield a P/O ratio of 2.5; those moved in by the glycerol 3-phosphate shuttle enter at CoQ and give a P/O ratio of 1.5.
REGULATION OF OXIDATIVE PHOSPHORYLATION::
Oxidative Phosphorylation Is Regulated by Cellular Energy Needs
The intracellular concentration of ADP is one measure of the energy status of cells. Another, related measure is the mass-action ratio of the ATP-ADP system, [ATP]/([ADP][Pi]).
An Inhibitory Protein Prevents ATP Hydrolysis during Ischemia
We have already encountered ATP synthase as an ATPdriven proton pump, catalyzing the reverse of ATP synthesis.
When a cell is ischemic (deprived of oxygen), as in a heart attack or stroke, electron transfer to oxygen ceases, and so does the pumping of protons. The proton-motive force soon collapses.
Under these conditions, the ATP synthase could operate in reverse, hydrolyzing ATP to pump protons outward and causing a disastrous drop in ATP levels.
This is prevented by a small (84 amino acids) protein inhibitor, IF1, which simultaneously binds to two ATP synthase molecules, inhibiting their ATPase activity.
IF1 is inhibitory only in its dimeric form, which is favored at pH lower than 6.5.
In a cell starved for oxygen, the main source of ATP becomes glycolysis, and the pyruvic or lactic acid thus formed lowers the pH in the cytosol and the mitochondrial matrix.
UNCOUPLED MITOCHONDRIA IN BROWN FAT PRODUCE HEAT
There is a remarkable and instructive exception to the general rule that respiration slows when the ATP supply is adequate.
Most newborn mammals, including humans, have a type of adipose tissue called brown fat in which fuel oxidation serves not to produce ATP but to generate heat to keep the newborn warm.
This specialized adipose tissue is brown because of the presence of large numbers of mitochondria and thus large amounts of cytochromes, whose heme groups are strong absorbers of visible light.
The mitochondria of brown fat are like those of other mammalian cells in all respects, except that they have a unique protein in their inner membrane. Thermogenin, also called the uncoupling protein , provides a path for protons to return to the matrix without passing through the FoF1 comp.
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