8.4 Other Functions of Nucleotides

Nucleotides carry chemical energy in cells

ATP is adenine plus ribose plus three phosphates, alpha, beta and gamma
The ribose 5-prime oxygen to alpha phosphate is a phosphoester, and alpha-beta and beta-gamma are phosphoanhydride bonds
ATP exists as several species including Mg2+ complexes, and enzymes distinguish between them
The phrase high-energy phosphate bond means only that hydrolysis has a large negative free energy
ATP plus water to ADP plus Pi has ΔG°′ near -30.5 kJ/mol
The actual ΔG depends on substrate and product concentrations, Mg2+, pH and ionic strength
Glucose phosphorylation at ΔG°′ near +13.8 kJ/mol proceeds through hexokinase with ATP, giving a combined ΔG°′ near -16.7 kJ/mol
Coupling works through a shared intermediate and an enzyme reaction, not by adding free energies in one vessel
ΔG = ΔG°′ + RT ln Q, where Q for ATP hydrolysis is [ADP][Pi] divided by [ATP]
Example: 3.0, 0.50 and 2.0 mM give Q near 3.33e-4 and ΔG near -50.3 kJ/mol
ATP to AMP plus PPi differs from ATP to ADP plus Pi, and the following PPi hydrolysis makes the overall direction favourable
GTP serves translation and G protein switching, UTP forms activated sugar precursors and CTP serves lipid synthesis
The adenylate energy charge is ([ATP] + 0.5[ADP]) divided by the adenylate total, ranging from 0 to 1
Example: 3.0, 0.8 and 0.2 mM give 0.85 while 1.5, 1.5 and 1.0 mM give 0.5625
Energy charge summarises an instant of energy status and does not replace the actual flux of every pathway

표 8.4-1Representative functions of nucleotides

Figure 8-67Overview of four functions in which nucleotides take part; the categories are not mutually exclusive

Figure 8-68Base, sugar and phosphate composition of ATP with its bond types distinguished

Figure 8-69An enzyme changes the activation barrier but not the ΔG between reactants and products

Figure 8-70Thermodynamic addition of glucose phosphorylation and ATP hydrolysis, using standard values

Figure 8-71Calculated ΔG of ATP hydrolysis as the ATP to ADP ratio changes; Pi 2 mM, 25 degrees Celsius

Figure 8-72Adenylation of ATP linked to pyrophosphate hydrolysis

Figure 8-73Nucleoside triphosphates other than ATP and their representative functions

Figure 8-74Energy charge against composition for three conditions with the same total adenylate

Figure 8-75Adenylate kinase and the link between AMP and ATP signalling

Adenine nucleotides are components of many coenzymes

Coenzymes are organic molecules that assist catalysis by carrying electrons, hydrogen or acyl groups
NAD+, NADP+, FAD and coenzyme A differ in structure and role yet all contain an adenosine nucleotide portion
The adenosine part mainly supports binding and recognition while the reactive centre is the nicotinamide, flavin or thiol
NAD+ accepts a hydride from substrate and becomes NADH
NADP+ carries an extra phosphate on the ribose 2-prime at the adenosine end, and that phosphate does not take part in the redox step
NADH and NADPH absorb at 340 nm while the oxidised forms absorb little there
Because 340 nm is ultraviolet, equipment and cuvettes capable of UV detection are required
Example: A340 of 0.622 with a 1.00 cm path and epsilon 6.22 gives 0.100 mM, and a fourfold diluted sample corresponds to 0.400 mM
FAD carries out two-electron, two-proton reduction through the flavin ring and can also occupy a semiquinone state
FAD often acts as a tightly bound prosthetic group
Coenzyme A carries acyl groups as thioesters through its terminal SH
Following NADH absorbance cannot monitor every reaction, only those in which it participates

표 8.4-2Representative coenzymes that contain adenosine

Figure 8-76Adenosine-related structures in NAD(P), FAD and CoA with their different reactive centres

Figure 8-77Hydride transfer from substrate to NAD+ forming NADH, simplified

Figure 8-79Typical metabolic roles of NAD(H) and NADP(H) compared

Figure 8-78Theoretical straight-line relation between NADH concentration and absorbance at 340 nm

Figure 8-80Oxidation states of the flavin coenzyme, with protein-dependent species simplified

Figure 8-81Thiol group of coenzyme A and the thioester bond of acetyl-CoA

Some nucleotides act as regulatory molecules

ATP is a phosphate donor and also binds allosteric sites on some regulatory enzymes
PFK-1 uses ATP as a substrate yet is inhibited through a separate regulatory effect when ATP is plentiful
AMP signals metabolic status and AMPK senses AMP, ADP and ATP ratios together with phosphorylation state
A GPCR activates G-alpha-s, which stimulates adenylyl cyclase to make cAMP and PPi
cAMP regulates PKA, EPAC and some ion channels
With dc/dt = p - kc the steady state is p/k, and after the stimulus ends the level decays exponentially with half-life ln2/k
Example: k of 0.14 per minute gives a half-life near 4.95 minutes, and 1.5 micromolar falls to about 0.370 micromolar after 10 minutes
Guanylyl cyclase makes cGMP from GTP, and nitric oxide activates the soluble form
cGMP acts through PKG and some ion channels to change vascular smooth muscle tone
Phosphodiesterases terminate the signal by converting cAMP and cGMP to 5-prime AMP and 5-prime GMP

표 8.4-3cAMP and cGMP compared

Figure 8-82Substrate and regulatory roles of ATP compared with AMPK control under energy stress

Figure 8-83Basic path of cAMP generation, target regulation and removal by phosphodiesterase

Figure 8-84Simulated cAMP time course with production 0.28 per minute, decay constant 0.14 per minute and stimulus ending at 10 minutes

Figure 8-85Representative intracellular signalling through the NO, soluble guanylyl cyclase and cGMP pathway

Adenine nucleotides also act as signals

Cells can release ATP through vesicular secretion and other routes
Extracellular ATP activates P2X ligand-gated ion channels or P2Y G-protein-coupled receptors
ADP is especially important in platelet P2Y12 signalling
CD39 degrades ATP and ADP while CD73 converts AMP to adenosine
Adenosine lacks phosphate and acts on P1 receptors as a nucleoside
Degradation therefore creates a new signal rather than only removing one
For a single site, occupancy theta = [L] divided by Kd plus [L], and theta is 0.5 when [L] equals Kd
Fifty percent occupancy does not mean fifty percent response, since spare receptors, amplification, desensitisation and ligand degradation shift the curve
Example: Kd 10 micromolar with 5 micromolar ligand gives theta of 0.333
ATP, ADP and AMP are nucleotides whereas adenosine is a nucleoside
cAMP acts as an intracellular second messenger and extracellular ATP as a receptor ligand, so both site and receptor differ

표 8.4-4Core questions about nucleotide functions

Figure 8-86Functional comparison of extracellular nucleotides at P2X, P2Y and adenosine P1 receptors

Figure 8-87Stepwise conversion of external ATP to ADP, AMP and adenosine, and the different receptor signals

Figure 8-88Simple occupancy calculation for two assumed Kd values; it does not represent the size of a cellular response

References

**표 8.4-1**Representative functions of nucleotides
Molecule	Representative reaction or function	Point to distinguish
ATP	Phosphate transfer, motor proteins, adenylation	Can give ADP or AMP
GTP	Translation steps, GTP-binding protein switches	Hydrolysis is coupled to switching
UTP	Formation of activated sugar precursors such as UDP-glucose	Sugar carriage is a function of the UDP-sugar
CTP	Lipid synthesis through CDP-activated intermediates	Used depending on enzyme and pathway

**표 8.4-2**Representative coenzymes that contain adenosine
Coenzyme	Reactive centre or carrier	Main role	Related vitamin
NAD+ and NADH	Nicotinamide, hydride	Oxidation and reduction in catabolism	Niacin, B3
NADP+ and NADPH	Nicotinamide, hydride	Reductive biosynthesis and antioxidant defence	Niacin, B3
FAD and FADH2	Flavin, electrons and hydrogen	Enzyme-bound oxidation and reduction	Riboflavin, B2
CoA and acyl-CoA	Terminal SH, acyl groups	Carriage of acetyl and fatty acyl groups	Pantothenate, B5

**표 8.4-3**cAMP and cGMP compared
Feature	cAMP	cGMP
Direct precursor	ATP	GTP
Synthesising enzyme	Adenylyl cyclase	Guanylyl cyclase
Main protein targets	PKA and EPAC	PKG
Termination	Phosphodiesterase gives 5-prime AMP	Phosphodiesterase gives 5-prime GMP
Example function	Metabolic and gene expression control	Smooth muscle relaxation and sensory signalling

**표 8.4-4**Core questions about nucleotide functions
Question	Short answer	Example
How do nucleotides drive work?	They couple hydrolysis or group transfer to a reaction	ATP to ADP, ATP to AMP plus PPi
What does adenine do in coenzymes?	It is a common structural element that aids recognition	NAD(H), FAD(H2), CoA
How is energy status sensed?	ATP, ADP and AMP ratios act on regulatory enzymes	AEC, AMPK
How do nucleotides act as intracellular signals?	Cyclic nucleotides regulate their targets	cAMP-PKA, cGMP-PKG
How do extracellular nucleotides act?	They become receptor ligands and are degraded to change the signal	P2X, P2Y, CD39, CD73, P1

**Figure 8-67**Overview of four functions in which nucleotides take part; the categories are not mutually exclusive

**Figure 8-68**Base, sugar and phosphate composition of ATP with its bond types distinguished

**Figure 8-69**An enzyme changes the activation barrier but not the ΔG between reactants and products

**Figure 8-70**Thermodynamic addition of glucose phosphorylation and ATP hydrolysis, using standard values

**Figure 8-71**Calculated ΔG of ATP hydrolysis as the ATP to ADP ratio changes; Pi 2 mM, 25 degrees Celsius

**Figure 8-72**Adenylation of ATP linked to pyrophosphate hydrolysis

**Figure 8-73**Nucleoside triphosphates other than ATP and their representative functions

**Figure 8-74**Energy charge against composition for three conditions with the same total adenylate

**Figure 8-75**Adenylate kinase and the link between AMP and ATP signalling

**Figure 8-76**Adenosine-related structures in NAD(P), FAD and CoA with their different reactive centres

**Figure 8-77**Hydride transfer from substrate to NAD+ forming NADH, simplified

**Figure 8-79**Typical metabolic roles of NAD(H) and NADP(H) compared

**Figure 8-78**Theoretical straight-line relation between NADH concentration and absorbance at 340 nm

**Figure 8-80**Oxidation states of the flavin coenzyme, with protein-dependent species simplified

**Figure 8-81**Thiol group of coenzyme A and the thioester bond of acetyl-CoA

**Figure 8-82**Substrate and regulatory roles of ATP compared with AMPK control under energy stress

**Figure 8-83**Basic path of cAMP generation, target regulation and removal by phosphodiesterase

**Figure 8-84**Simulated cAMP time course with production 0.28 per minute, decay constant 0.14 per minute and stimulus ending at 10 minutes

**Figure 8-85**Representative intracellular signalling through the NO, soluble guanylyl cyclase and cGMP pathway

**Figure 8-86**Functional comparison of extracellular nucleotides at P2X, P2Y and adenosine P1 receptors

**Figure 8-87**Stepwise conversion of external ATP to ADP, AMP and adenosine, and the different receptor signals

**Figure 8-88**Simple occupancy calculation for two assumed Kd values; it does not represent the size of a cellular response