High Energy Bonds

The structure of ATP is shown below at right (see also p. 566). Anhydride bonds (in red) link the terminal phosphates.

Phosphoanhydride bonds (formed by splitting out water between two phosphoric acids or between a carboxylic acid and a phosphoric acid) tend to have a large negative DG of hydrolysis, and are thus said to be "high energy" bonds. It is important to realize that the bond energy is not necessarily high, just the free energy of hydrolysis.

"High energy" bonds are often represented by the " ~ " symbol (squiggle), with ~P representing a phosphate group with a high free energy of hydrolysis.

Compounds with "high energy" bonds are said to have high group transfer potential. For example, P_i may be spontaneously removed from ATP for transfer to another compound (e.g., to a hydroxyl group on glucose).

Potentially two "high energy" bonds can be cleaved, as two phosphates are released by hydrolysis from ATP (adenosine triphosphate), yielding ADP (adenosine diphosphate), and ultimately AMP (adenosine monophosphate). This may be represented as follows (omitting waters of hydrolysis):

• AMP~P~P ® AMP~P + P_i (ATP ® ADP + P_i)
• AMP~P ® AMP + P_i (ADP ® AMP + P_i)

Alternatively, as discussed above:

• AMP~P~P ® AMP + P~P_i (ATP ® AMP + PP_i)
• P~P ® 2 P_i

ATP often serves as an energy source. Hydrolytic cleavage of one or both of the "high energy" bonds of ATP is coupled to an energy-requiring (non-spontaneous) reaction, as in the examples presented above.

AMP functions as an energy sensor and regulator of metabolism. When ATP production does not keep up with needs, a higher portion of a cell's adenine nucleotide pool is in the form of AMP. AMP then stimulates metabolic pathways that produce ATP.

• Some examples of this role involve direct allosteric activation of pathway enzymes by AMP. (E.g., activation of the Glycogen Phosphorylase enzyme by AMP will be discussed later.)
• Some regulatory effects of AMP are mediated by the enzyme AMP-Activated Protein Kinase. (For example the role of AMP-Activated Protein Kinase in stimulation of fatty acid catabolism by AMP will be discussed later.)

Artificial ATP analogs have been designed that are resistant to cleavage of the terminal phosphate by hydrolysis, e.g., AMPPNP, depicted at right. Such analogs have been used to study the dependence of coupled reactions on ATP hydrolysis. In addition, they have made it possible to crystallize an enzyme that catalyzes ATP hydrolysis with an ATP analog at the active site.

A reaction that is important for equilibrating ~P among adenine nucleotides within a cell is that catalyzed by Adenylate Kinase:

ATP + AMP «2 ADP

The Adenylate Kinase reaction is also important because the substrate for ATP synthesis, e.g., by the mitochondrial ATP Synthase, is ADP, while some cellular reactions dephosphorylate ATP all the way to AMP.

The enzyme Nucleoside Diphosphate Kinase (NuDiKi) equilibrates ~P among the various nucleotides that are needed, e.g., for synthesis of DNA and RNA. NuDiKi catalyzes reversible reactions such as:

ATP + GDP «ADP + GTP, ATP + UDP «ADP + UTP, etc.

Many organisms store energy as inorganic polyphosphate, a chain of many phosphate residues linked by phosphoanhydride bonds. It may be represented as: P~P~P~P~P... Hydrolysis of P_i residues from polyphosphate may be coupled to energy-dependent reactions. Depending on the organism or cell type, inorganic polyphosphate may have additional functions. For example, it may serve as a reservoir for P_i, a chelator of metal ions, a buffer, or a regulator.

Why do phosphoanhydride linkages have a high free energy of hydrolysis? Contributing factors for ATP and PP_i are thought to include:

• Resonance stabilization of the products of hydrolysis exceeds resonance stabilization of the compound itself. See Fig. 16-22 p. 568.
• Electrostatic repulsion between negatively charged phosphate oxygens favors separation of the phosphates.

Phosphocreatine (also called creatine phosphate), another compound with a "high energy" phosphate linkage, is used in nerve and muscle cells for storage of ~P bonds. Creatine Kinase catalyzes: phosphocreatine + ADP«ATP + creatine This is a reversible reaction, though the equilibrium constant slightly favors phosphocreatine formation. Phosphocreatine is produced when ATP levels are high. When ATP is depleted during exercise in muscle, phosphate is transferred from phosphocreatine to ADP, to replenish ATP.

Phosphoenolpyruvate (PEP), involved in production of ATP in Glycolysis, has a larger negative DG of phosphate hydrolysis than ATP. Removal of phosphate from the ester linkage in PEP is spontaneous because the enol product spontaneously converts to a ketone.

The ester linkage in PEP is an exception. Generally phosphate esters, formed by splitting out water between a phosphoric acid and a hydroxyl group, have a low but negative DG of hydrolysis. Examples, shown below, include:

the linkage between the first phosphate and the ribosehydroxyl of ATP. the linkage between phosphate and a hydroxyl group in glucose-6-phosphate or glycerol-3-phosphate. the linkage between phosphate and the hydroxyl group of an amino acid residue in a protein (serine, threonine or tyrosine). Regulation of proteins by phosphorylation and dephosphorylation will be discussed later. An example mentioned above is AMP-Activated Protein Kinase.

ATP has special roles in energy coupling and phosphate transfer. The free energy of hydrolysis of phosphate from ATP is intermediate among the examples listed in the table below (more complete table p. 566). ATP can thus act as a phosphate donor, and ATP can be synthesized by transfer of phosphate from other compounds, such as phosphoenolpyruvate (PEP).

Compound	DG^o' of phosphate hydrolysis (kJ/mol)
Phosphoenolpyruvate (PEP)	- 61.9
Phosphocreatine	- 43.1
Pyrophosphate	- 33.5
ATP (to ADP)	- 30.5
Glucose-6-phosphate	- 13.8
Glycerol-3-phosphate	- 9.2

Some other "high energy" bonds: A thioester forms between a carboxylic acid and a thiol (SH) group, e.g., the thiol of coenzyme A (abbreviated CoA-SH). Thioesters are "high energy" linkages. In contrast to phosphate esters, thioesters have a large negative DG of hydrolysis.

The thiol of coenzyme A can react with a carboxyl group of acetic acid (yielding acetyl-CoA) or a fatty acid (yielding fatty acyl-CoA). The spontaneity of thioester cleavage is essential to the role of coenzyme A as an acyl group carrier. Like ATP, acyl-coenzyme A has a high group transfer potential.

Coenzyme Aincludes b-mercaptoethylamine, in amide linkage to the carboxyl group of the B vitamin pantothenate. The hydroxyl of pantothenate is in ester linkage to a phosphate of ADP-3'-phosphate. The functional group is the thiol (SH) of b-mercaptoethylamine.

3',5'-Cyclic AMP(abbreviated cAMP), shown at right and below, is used by cells as a transient signal. Adenylate Cyclase (Adenylyl Cyclase) catalyzes cAMP synthesis: ATP®cAMP + PPi. The reaction is highly spontaneous due to the production of PPi, which spontaneously hydrolyzes. Phosphodiesterase catalyzes catalyzes hydrolytic cleavage of one of the phosphate ester linkages (in red), convertingcAMP®5'-AMP. This is a highly spontaneous reaction, because cAMP is sterically constrained by having a phosphate with ester linkages to two hydroxyls of the same ribose. The lability of cAMP to hydrolysis makes it an excellent transient signal. Signal roles of cAMP will be discussed separately.

Explore at right the structure of cAMP. The coordinate file for this display was obtained using CHEM 3D with MM2 energy minimization. Right click to change the display to sticks or ball & stick. Drag the image to view the molecular conformation, and compare to the structure diagram above. Identify the bond that is cleaved when cAMP is hydrolyzed to AMP. Why is the cleavage of this bond by hydrolysis spontaneous?

C O N P H

Distinction between thermodynamics and kinetics: A high activation energy barrier usually causes hydrolysis of a "high energy bond" to be very slow in the absence of an enzyme catalyst. This " kinetic stability " is essential to the role of ATP and other compounds with ~ bonds. If ATP would rapidly hydrolyze in the absence of a catalyst, it could not serve its important roles in energy metabolism and phosphate transfer. Phosphate is removed from ATP only when the reaction is coupled via enzyme catalysis to some other reaction useful to the cell, such as transport of an ion, phosphorylation of glucose, or regulation of an enzyme by phosphorylation of a serine residue.

Problems relating to bioenergetics are on the page with potential test questions.

Oxidation & reduction will be covered in more detail later. A brief introduction to selected topics will be presented here. The evolution of photosynthesis, and the generation of the oxygen that is now plentiful in our environment, allowed development of metabolic pathways that derive energy from transfer of electrons from various reductants ultimately to molecular oxygen.

Oxidation of an iron atom involves loss of an electron (to some acceptor atom):

Fe⁺⁺ (reduced)®Fe⁺⁺⁺ (oxidized) + e^-

For a carbon compound, increased oxidation means increased number of C-O bonds. Since electrons in a C-O bond are associated more with the oxygen, the C becomes relatively electron deficient as you go from hydrocarbon to CO2. Oxidation of carbon is spontaneous (energy yielding).

Two important electron carriers in metabolism are NAD^+ and FAD. NAD^+ (Nicotinamide Adenine Dinucleotide) functions as an electron acceptor in catabolic pathways. The nicotinamide ring of NAD^+, which is derived from the vitamin niacin,accepts 2 e^- and one H^+ (a hydride) in going to the reduced state, as NAD^+ becomes NADH. See also p. 461 & 555. NADP^+/NADPH is similar, except for an additional phosphate esterified to a hydroxyl group on the adenosine ribose. NADPH functions as an electron donor in synthetic pathways.

The electron transfer reaction may be summarized as: NAD^+ + 2 e^- + H^+ «NADH It may also be written as: NAD^+ + 2 e^- + 2H^+ «NADH + H^+

FAD (F lavin A denine D inucleotide) also functions as an electron acceptor. The portion of FAD that undergoes reduction/oxidation is the dimethylisoalloxazine ring, derived from the vitamin riboflavin. See p. 556.

FAD normally accepts 2 e^- and 2 H⁺ in going to its reduced state:
FAD + 2 e^- + 2 H⁺ «FADH₂

• NAD⁺ is a coenzyme, that reversibly binds to enzymes.
• FAD is a prosthetic group, that usually remains tightly bound at the active site of an enzyme.

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