Biological oxidation reactions serve two functions, as described in the previous chapter. Oxidation of organic molecules can produce new molecules with different properties. For example, increases in solubility is observed on hydroxylation of aromatic substrates by cytochrome P450. Likewise, amino acids can by oxidized to produce neurotransmitters. Most biological oxidation reactions occur, however, to produce energy to drive thermodynamically unfavored biological processes such as protein and nucleic acid synthesis, or motility. Chemical potential energy is not just released in biological oxidation reactions. Rather, it is transduced into a more useful form of chemical energy in the molecule ATP (adenosine triphosphate). This chapter will discuss the properties that make ATP so useful biologically, and how exergonic biological oxidation reactions are coupled to the synthesis of ATP.
PROPERTIES OF ATP
ATP contains two phosphoanhydride bonds (connecting the 3 phosphates together) and one phosphoester bond (connecting a phosphate to the ribose ring). The pKa's for the reactions HATP3- ---> ATP4- + H+ and HADP2- ---> ADP3- + H+ are about 7.0, so the overall charges of ATP and ADP at physiological pH are -3.5 and -2.5, respectively. Each of the phosphorous atoms are highly electrophilic and can react with nucleophiles like the OH of water or an alcohol. As we discussed earlier, anhydrides are thermodynamically more reactive than esters which are more reactive than amides. The large negative ΔGo (-7.5 kcal/mol) for the hydrolysis (a nucleophilic substitution reaction) of one of the phosphoanhydride bonds can be attributed to a relative destabilization of the reactants (ATP and water) and relative stabilization of the products (ADP = Pi). Specifically
- The reactants can not be stabilized to the same extent as products by resonance due to competing resonance of the bridging anhydride O's.
- The charge density on the reactants is greater than that of the products
- Theoretical studies show that the products are more hydrated than the reactants.
The ΔGo for hydrolysis of ATP is dependent on the divalent ion concentration and pH, which affect the the stabilization and the magnitude of the charge states of the reactants and products.
Figure: STRUCTURE AND HYDROLYSIS OF ATP
Carboxylic acid anhydrides are even more unstable to hydrolysis than ATP (-20 kcal/mol), followed by mixed anhydrides (-12 kcal/mol), and phosphoric acid anhydrides (-7.5 kcal/mol). These molecules are often termed "high energy" molecules, which is somewhat of a misnomer. They are high energy only in relation to the energy of their cleavage products, such that the reaction proceeds with a large negative ΔGo.
Figure: HIGH ENERGY MOLECULES
How can ATP be used to drive thermodynamically unfavored reaction? First consider how the hydrolysis of a carboxylic acid anhydride, which has a ΔGo = -12.5 kcal/mol can drive the synthesis of a carboxylic acid amide, with a Δ Go = + 2-3 kcal/mol. The link below shows the net reaction, (anhydride + amine --> amide + carboxylic acid), which can be broken into two reactions: hydrolysis of the anhydride, and the synthesis of the amide.
Now consider the reaction of glucose + Pi to form glucose-6-P. In this reaction a phosphoester is formed, so the reaction would proceed with a positive ΔGo = 3.3. Now if ATP was used to transfer the terminal (gamma) phosphate to glucose to form Glc-6-P, the reaction proceeds with a ΔGo = -4 kcal/mol. This can be calculated since ΔG is a state function and is path independent. Adding the reactions and the ΔGo's for glucose + Pi ------> glucose-6-P and
ATP + H2O -----> ADP + Pi gives the resultant reaction and ΔGo,
glucose + ATP -----> Glucose-6-P + ADP, ΔGo = -4.
In most biological reactions using ATP, the terminal P of ATP is transferred to a substrate using an enzyme called a kinase. Hence, hexokinase transfers the gamma phosphate from ATP to a hexose sugar. Protein kinase is an enzyme which transfers the gamma phosphate to a protein substrate.
ATP is also used to drive peptide bond (amide) synthesis during protein synthesis. From an energetic point of view, anhydride cleavage can provide the energy for amide bond formation. Peptide bond synthesis is cells is accompanied by cleavage of both phosphoanhydride bonds in ATP in a complicated set of reactions that is catalyzed by ribosomes in the cells. (This topic is considered in depth in molecular biology courses). The figure below is a grossly simplified mechanism of how peptide bond formation can be coupled to ATP cleavage.
Phosphorylation reactions using ATP are really nucleophilic substitution reactions which proceed through a pentavalent intermediate. The rest of the ATP molecule is then considered the leaving group, which could be theoretically ADP or AMP as well. If water is the nucleophile, the reaction is also a hydrolysis reaction. These reactions are also called phosphoryl transfer reactions.
One last note. ATP exists in cells as just one member of a pool of adenine nucleotides which consists of not only ATP, but also ADP and AMP (along with Pi). These constituents are readily interconvertible. We actually break down an amount of ATP each day equal to about our body weight. Likewise we make about the same amount from the turnover products. When energy is needed, carbohydrates and lipids are oxidized and ATP is produced, which can then be immediately used for motility, biosynthesis, etc. It is very important to realize that although ATP is converted to ADP in a thermodynamically spontaneous process, the process is kinetically slow without an enzyme. Hence ATP is stable in solution. However, its biological half-life is not long since it is used very quickly as described above. This recapitulates a theme we have seen before. Many reactions (like oxidation with dioxygen, denaturation of proteins in nonpolar solvent, and now ATP hydrolysis) are thermodynamically favored but kinetically slow. This kinetic slowness is a necessary but of course insufficient condition, for life.
Introduction to Active Transport
We have previously discussed how chemical potential energy in the form of reduced organic molecules can be transduced into the chemical potential energy of ATP. This ATP can be used to drive reductive biosynthesis and movement (from individual cells to whole organisms). ATP has two other significant uses in the cell.
Active Transport: Molecules must often move across membranes against a concentration gradient - from low to high chemical potential - in a process characterized by a positive ΔG. As protons could be "pumped" across the inner mitochondrial membrane against a concentration gradient, powered by the ΔG associated with electron transport (passing electrons from NADH to dioxygen), other species can cross membranes against a concentration gradient - a process called active transport - if coupled to ATP hydrolysis or the collapse of another gradient. This active transport is differentiated from facilitated diffusion we studied earlier, which occurred down a concentration gradient across the membrane. Many such species must be transported into the cell or into intracellular organelles against a concentration gradient!
Signal Transduction: All cells must know how to respond to their environment. They must be able to divide, grow, secrete, synthesize, degrade, differentiate, cease growth, and even die when the appropriate signal is given. This signal invariably is a molecule which binds to a receptor, typically on the cell surface. (Exceptions include light transduction in retinal cells when the signal is a photon, and lipophilic hormones which pass through the membrane.) Binding is followed by shape changes in transmembrane protein receptors which effectively transmits the signal into the cytoplasm. We will discuss two main types of signal transduction pathways:
- nerve conduction, in which a presynaptic neuron releases a neurotransmitter causing a postsynaptic neuron to "fire";
- signaling at the cell surface which leads to activation of kinases within the cytoplasm;
We will discuss signal transduction in the final two chapters.
For active transport to occur, a membrane receptor is required which recognizes the ligand to be transported. Of major interest to us, however, is the energy source used to drive the transport against a concentration gradient. The biological world has adapted to use almost any source of energy available.
Energy released by oxidation: We have already encountered the active transport of protons driven by oxidative processes. In electron transport in respiring mitochondria, NADH is oxidized as it passes electrons to a series of mobile electron carriers (ubiquione, cytochrome C, and eventually dioxygen) using Complex 1, 3 and 4 in the inner membrane of the mitochondria. Somehow the energy lost in this thermodynamically favored process was coupled to conformational changes in the complex which caused protons to be ejected from the matrix into the inner membrane space. One can imagine a series of conformation-sensitive pKa changes in various side chains in the complexes which lead in concert to the vectorially discharge of protons.
ATP hydrolysis: One would expect that this ubiquitous carrier of free energy would by used to drive active transport. In fact, this is one of the predominant roles of ATP in the biological world. 70% of all ATP turnover in the brain is used for the creation and maintenance of a Na and K ion gradient across nerve cell membranes using the membrane protein Na+/K+ ATPase.
Light: Photosynthetic bacteria have a membrane protein called bacteriorhodopsin which contains retinal, a conjugated polyene derived from beta-carotene. It is analogous to the visual pigment protein rhodopsin in retinal cells. Absorption of light by the retinal induces a conformation changes in the retinal and protein, which leads to vectorial discharge of protons ;
Collapse of an ion gradient: The favorable collapse of an ion gradient can be used to drive the transport of a different species against a concentration gradient. We have already observed that collapse of a proton gradient across the inner mitochondria membrane (through FoF1ATPase) can drive the thermodynamically unfavored synthesis of ATP. Collapse of a proton gradient provides a proton-motive force which can drive the active transport of sugars. Likewise a sodium-motive force can drive active transport of metal ions. Since the energy to make the initial ion gradients usually comes from ATP hydrolysis, ATP indirectly powers the transport of the other species against a gradient.
Often times, transport of one species is coupled to transport of another. If the species are charged, a net change in charge across the membrane may occur. Several terms are used to describe various types of transport:
- symport - two species are cotransported in the same direction by the same transport protein
- antiport - two species are cotransported in opposite directions by the same transport protein
- electrogenic - a net electrical imbalance is generated across the membrane by symport or antiport of charged species
- electroneutral - no net electrical imbalance is generated across the membrane by symport or antiport of charged species