L01 Chemical Logic of Metabolism

一、A First Look at Metabolism

Different Kinds of Organisms

1. Two Major Categories

Metabolism can be subdivided into two major categories:

1. Catabolism（分解代谢） - those processes in which complex substances are degraded to simpler molecules.

2. Anabolism （合成代谢）- those processes concerned primarily with the synthesis of complex organic molecules.

Catabolism is generally accompanied by the net release of chemical energy.

Anabolism requires a net input of chemical energy.

These two sets of reactions are coupled together by ATP.

2. Source of The Fuel Molecules

A fundamental distinction among organisms lies in the source of their fuel molecules.

1. Autotrophs（自养生物） (from Greek, “self-feeding”) synthesize glucose and all of their other organic compounds from inorganic carbon, supplied as CO2.

2. Heterotrophs（异养生物） (“feeding on others”) can synthesize their organic metabolites only from other organic compounds, which they must therefore consume.

A primary difference between plants and animals is that plants are autotrophs and animals are heterotrophs.

3. Need of Oxygen

Microorganisms show adaptability (适应性) with respect to their ability to survive in the absence of oxygen.

Virtually all multicellular organisms and many bacteria are strictly aerobic（需氧的，有氧的） organisms; they depend absolutely upon respiration, the coupling of energy generation to the oxidation of nutrients by oxygen.

By contrast, many microorganisms either can, or must, grow in anaerobic（无氧;厌氧菌的） environments, deriving their metabolic energy from processes that do not involve molecular oxygen.

Three Stages

Both catabolic and anabolic pathways occur in three stages of complexity:

1. Stage 1: the interconversion of polymers and complex lipids with monomeric intermediates
2. Stage 2: the interconversion of monomeric sugars, amino acids, and lipids with still simpler organic compounds
3. Stage 3: the ultimate degradation to, or synthesis from, inorganic compounds, including CO2, H2O and NH3.

Brief Overview of Metabolism

A brief overview of metabolism:

We shall also use the terms intermediary metabolism, energy metabolism, and central pathways

1. Intermediary metabolism (中间代谢) comprises all reactions concerned with

   1. storing and generating metabolic energy and
   2. with using that energy in biosynthesis of low-molecular-weight compounds (intermediates) and
   3. energy storage compounds.
   • Not included are nucleic acid and protein biosynthesis from monomeric precursors. The reactions of intermediary metabolism can be thought of as those that do not involve a nucleic acid template because the information needed to specify each reaction is provided within the structure of the enzyme catalyzing that reaction
   • 可以将中间代谢的反应视为不涉及核酸模板的反应，因为在催化该反应的酶结构中提供了指定每种反应所需的信息。 可以将中间代谢的反应视为不涉及核酸模板的反应，因为在催化该反应的酶结构中提供了指定每种反应所需的信息。

2. Energy metabolism (能量代谢) is that part of intermediary metabolism consisting of pathways that store or generate metabolic energy.

3. Central pathways of metabolism (代谢的中心途径) are substantially the same in many different organisms,

   • and they account for relatively large amounts of mass transfer and energy generation within a cell; they are the quantitatively major pathways
   • The central pathways involve the oxidation of fuel molecules and the synthesis of small biomolecules from the resulting fragments; these pathways are found in all aerobic organisms

Intermediary metabolism（中间代谢） refers primarily to the biosynthesis, utilization, and degradation of low-molecular-weight compound (intermediates).

二、Freeways on the Metabolic Road Map

The first pathway that we study is glycolysis（糖酵解）, a stage 2 pathway for degradation of carbohydrates, in either aerobic or anaerobic cells.

The major input to glycolysis is glucose, usually derived from either energy-storage polysaccharides or dietary carbohydrates.

This pathway leads to pyruvate（丙酮酸）, a three-carbon α-keto acid.

Anaerobic organisms reduce pyruvate to a variety of products, for example lactate（乳酸）, or ethanol plus carbon dioxide.

These processes are called fermentations.

In oxidative metabolism (respiration), the major fate of pyruvate is its oxidation to a metabolically activated two-carbon fragment, acetyl-CoA.

乙酰辅酶A

The two carbons in the acetyl group then undergo oxidation in the citric acid cycle.

In aerobic organisms the citric acid cycle is the principal stage 3 pathway.

This cyclic pathway accepts simple carbon compounds, derived not only from carbohydrate but also from lipid or protein, and oxidizes them to CO2.

Using the freeway analogy again, we will see that numerous on-ramps（引导滑道） from the highways and byways of stage 1 and stage 2 metabolism lead to the citric acid cycle. In fact, all catabolic pathways converge at this point.

Overview of Pyruvate Oxidation and the Citric Acid Cycle

1. Citric Acid Cycle

The fate of carbon in the citric acid cycle:

Note that these departing CO2 groups derive from the two oxaloacetate carboxyl groups（草酰乙酸羧基组） that were incorporated as acetyl-CoA in earlier turns of the cycle.

Oxidative reactions of the citric acid cycle generate reduced electron carriers（还原电子载体） whose reoxidation drives ATP biosynthesis, primarily through processes in the mitochondrial respiratory chain—electron transport and oxidative phosphorylation（氧化磷酸化）.

The mitochondrial membrane uses oxidative energy to maintain a transmembrane gradient of hydrogen ion concentration (the protonmotive force（质子驱动力）), and discharge of this electrochemical potential energy powers the synthesis of ATP from ADP + Pi.

Oxidative Phosphorylation

Chemiosmotic（化学渗透的） coupling of electron transport and ATP synthesis:

Protons are pumped by complexes I, III, and IV as electrons flow through the complexes, generating an electrochemical gradient across the membrane (protonmotive force, pmf).

Proton re-entry to the matrix, through the F0 channel of ATP synthase (complex V), provides the energy to drive ATP synthesis.

Chemiosmotic coupling refers to the use of a transmembrane proton gradient to drive endergonic（吸能的） processes like ATP synthesis.

Overview of Metabolism

Overview of metabolism:

Shown here are the central metabolic pathways and some key intermediates.

Catabolic pathways (red) proceed downward and

Anabolic pathways (blue) proceed upward.

Note the three stages of metabolism.

1. Initial Phase: Glycolysis

The initial phase of carbohydrate catabolism: glycolysis:

Pyruvate either undergoes reduction in fermentation reactions or enters oxidative metabolism (respiration) via conversion to acetyl-CoA

2. Oxidative metabolism

Oxidative metabolism includes

• Pyruvate oxidation

• The citric acid cycle

• Electron transport

• Oxidative phosphorylation

  Pyruvate oxidation supplies acetyl-CoA to the citric acid cycle.

3. Carbohydrate anabolism

Biosynthesis of carbohydrates includes gluconeogenesis（糖再生） and polysaccharide synthesis（多糖合成）.

4. Photosynthesis

(+) 5. Distinct Pathways for Biosynthesis and Degradation

It is important to realize that in these cases the opposed pathways are quite distinct from one another

They may share some common intermediates or enzymatic reactions, but they are separate reaction sequences, regulated by distinct mechanisms and with different enzymes catalyzeing their regulated reactions. They may even occur in separate cellular compartments

Biosynthetic and degradative pathways are never simple reversals of on another, even though they often begin and end with the same metabolites

Thus, opposed biosynthetic and degradative pathways must both be exergonic, and thus unidirectional, in their respective directions.

Using separate pathways for the biosynthetic and degradative processes is crucial for control, so conditions that activate one pathway tend to inhibit the opposed pathway and vice versa.

Futile Cycle

Degradative and biosynthetic pathways are distinct for two reasons:

A pathway can be exergonic in only one direction

Pathways must be separately regulated to avoid futile cycles（无效循环）.

futile cycle: No useful work is done

Compartmentation and allosteric control of anabolic and catabolic processes prevent futile cycles, which simply waste energy.

A similar futile cycle could result from the interconversion of fructose-6-phosphate with fructose-1,6- bisphosphate in carbohydrate metabolism.

However, enzymes catalyzing both of the above reactions respond to allosteric effectors, such that one enzyme is inhibited by conditions that activate the other. This reciprocal control (相互控制) prevents the futile cycle from occurring, even though the two enzymes occupy the same cell compartment.

Therefore, it is more appropriate to call this situation—two seemingly opposed cellular reactions that are independently controlled— a substrate cycle. (底物循环)

底物循环（substrate cycle）。一对催化两个途径的中间代谢物之间循环的方向相反、代谢上不可逆的反应。有时该循环通过ATP的水解导致热能的释放。例如，葡萄糖+ATP=葡萄糖-6-磷酸+ADP与葡萄糖-6-磷酸+H2O=葡萄糖+Pi反应组成的循环反应，其净反应实际上是ATP+H2O=ADP+Pi。

三、Biochemical Reaction Types

Much of the chemistry of biological molecules is the chemistry of the carbonyl group because the vast majority of biological molecules contain them.

Most of the chemistry of carbonyl groups involves nucleophiles（亲核试剂） (abbreviated “Nu:”) and electrophiles（亲电试剂）.

Recall that a nucleophile is a “nucleus-loving” substance with a negatively polarized, electron-rich atom that can form a bond by donating a pair of electrons to an electron-poor atom.

An electrophile is an “electron-loving” substance with a positively polarized, electron-poor atom that can form a bond by accepting a pair of electrons from an electron-rich atom.

1. 常见的Electrophiles: Carbonyl carbons (羰基碳原子), protonated imines (质子化亚胺), phosphate groups, and protons.
2. 常见的Nucleophiles: Oxyanions (含氧阴离子 ) (e.g., hydroxide ion, alkoxides (醇盐), or ionized carboxylates (离子化羧化物)), thiolates (硫醇盐) (deprotonated sulfhydryls (脱质子磺酸, sulfhydryls is the -SH )), carbanions (碳负离子), deprotonated amines, and the imidazole side chain of histidine (咪唑侧链组氨酸)

1. Nucleophilic substitution

In a nucleophilic substitution reaction, one nucleophile replaces a second nucleophile (the leaving group) on an -hybridized carbon atom. The leaving group develops a partial negative charge in the transition state, and the best leaving groups are those that are stable as anions.

Thus, halides（卤化物） and the conjugate bases of strong acids, such as the phosphate anion, are good leaving groups.

SN1 mechanism

The SN1 mechanism is a substitution reaction in organic chemistry. “SN“ stands for nucleophilic substitution and the “1” represents the fact that the rate-determining step is unimolecular.

SN1 (Substitution, Nucleophilic, Unimolecular)

SN2 mechanism

The SN2 ( (Substitution, Nucleophilic, Bimolecular) ) reaction (Substitution, Nucleophilic, Bimolecular)) is a type of reaction mechanism that is common in organic chemistry.

The attacking nucleophile approaches one side of the electrophilic center while the leaving group remains partially bonded to other side

acyl substitution reaction

An acyl substitution reaction mechanism involves a tetrahedral oxyanion reaction intermediate.

Nucleophilic acyl substitution describe a class of substitution reactions involving nucleophiles and acyl compounds. In this type of reaction, a nucleophile – such as an alcohol, amine, or enolate (烯醇化物) – displaces the leaving group of an acyl derivative – such as an acid halide, anhydride, or ester. The resulting product is a carbonyl-containing compound in which the nucleophile has taken the place of the leaving group present in the original acyl derivative.

Acyl substitutions occur most readily when the carbonyl carbon is bonded to an electronegative atom (such as O or N) or a highly polarizable atom (such as S)

2. Nucleophilic addition

The carbonyl carbon in aldehydes and ketones is bonded to atoms (C and H) that cannot stabilize a negative charge, and thus are not good leaving groups

These carbonyl groups typically undergo nucleophilic addition reactions, instead of substitution reactions.

Recall from Chapter 11 that an oxyanion intermediate is formed in the initial steps of peptide bond hydrolysis by the serine proteases.

The oxyanion intermediate then has several fates, depending on the nucleophile (Figure 12.7).

1. When the attacking nucleophile is a hydride ion (H:-), the oxyanion intermediate undergoes protonation to form an alcohol. Alcohols also result when the attacking nucleophile is a carbanion (R3C^-^) and this is one of the mechanisms that yield new C-C bonds.

2. When an oxygen nucleophile adds, such as an alcohol (ROH), the oxyanion intermediate undergoes proton transfer to yield a hemiacetal （半缩醛）. This reaction is the basis of ring formation in monosaccharides (Chapter 9).

3. Reaction with a second equivalent of alcohol gives an acetal. When the attacking nucleophile is a primary amine (R’ NH2) the oxyanion intermediate picks up a proton from the amino group, giving a carbinolamine, which loses water to form an imine (R2C = NR’).Imines (called Schiff bases) are common reaction intermediates in many biochemical reactions due to their ability to delocalize electrons.

3. Carbonyl condensation（羰基缩合）

Carbonyl condensation reactions:

These reactions are initiated by deprotonation of the weakly acidic hydrogen to give a resonance-stabilized enolate ion (top).

A carbonyl condensation reaction relies on the weak acidity of the $\alpha$ carbonyl hydrogen, producing a carbanion, which is in resonance with a nucleophilic enolate (烯醇化物) ion.

烯醇（Enol）指的是双键碳上连有羟基的一类化合物，其与羰基化合物成互变异构

The enolate ion, stabilized by resonance, nucleophilically adds to the electrophilic carbon of a second carbonyl, forming a new bond (Figure 12.8)

• In an alsdol condensation (醛醇缩合) (left side), the enolate adds to an aldehyde or ketone, yielding a β-hydroxycarbonyl product.
• In a Claisen condensation (right side), the enolate adds to an ester, yielding a β-keto product.

Aldol and Claisen condensations are both reversible

If the second carbonyl is an aldehyde or ketone (an aldol condensation), this nucleophilic addition produces an oxyanion intermediate, which is protonated to give a $\beta$-hydroxy carbonyl product.

If the second carbonyl is an ester (a Claisen condensation), the intermediate oxyanion expels the ester alkoxide (RO-) as the leaving group, giving a $\beta$-keto product

4. Eliminations

Elimination reactions can occur by several different mechanisms, but the most common one involves a carbanion intermediate.

The reactant is often a $\beta$-hydroxy carbonyl (where X=OH) in which the H atom to be removed is made more acidic by being adjacent to a carbonyl group. A base abstracts the proton to give a carbanion intermediate (resonance-stabilized with the enolate) that loses OH^-^ to form C=C the double bond. $\beta$-hydroxy carbonyl compounds are readily dehydrated via these $\alpha$,$\beta$-elimination reactions.

Example:

5. Oxidative and Reductions

Energy production in most cells involves the oxidation of fuel molecules such as glucose.

Oxidation-reduction, or redox, chemistry thus lies at the core of metabolism.

Redox reactions involve reversible electron transfer from a donor (the reductant) to an acceptor (the oxidant).

In this example, because the alcohol has lost a pair of electrons and two hydrogen atoms, this type of oxidation is called dehydrogenation（脱氢作用）, and enzymes that catalyze this reaction are called dehydrogenase .

A base abstracts the weakly acidic O-H proton, the electrons from that C=O bond move to form a bond, and the C-H bond is cleaved. The hydrogen and the electron pair (i.e., hydride, H-) add to NAD^+^ in a nucleophilic addition reaction, reducing it to NADH

The plus sign in NAD^+^ reflects the charge on the pyridine ring nitrogen in the oxidized form; this charge is lost as the electron pair moves through the ring onto this nitrogen

redox reactions are reversible, and dehydrogenases can catalyze the reductive direction as well.

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There are other, less common types of reactions in biochemical pathways, such as free radical reactions, but these five represent the basic toolkit that cells use to carry out the vast majority of their chemical transformations

四、Some Bioenergetic Considerations

Oxidation as a Metabolic Energy Source

Oxygen, the ultimate electron acceptor for aerobic organisms, is a strong oxidant; it has a marked tendency to attract electrons, becoming reduced in the process

(+)1. Energy Release in Small Increments

In catabolism of glucose, about 40% of the released energy is used to drive the synthesis of ATP from ADP and Pi.

A series of coupled oxidation–reduction reactions occurs, with the electrons passed to intermediate electron carriers such as NAD^+^ and FAD, and finally transferred to oxygen

Reactions 12.2 and 12.3 are examples of obligate-coupling stoichiometry. These are stoichiometric relationships fixed by the chemical nature of the process. Thus, the complete oxidation of glucose requires the transfer of 12 pairs of electrons from glucose to molecular oxygen, whether as a direct process, as in combustion, or via intermediate electron carriers, as in the biological process.

The transfer of electrons from these intermediate electron carriers to oxygen is catalyzed by the electron transport chain, or respiratory chain, and oxygen is called the terminal electron acceptor.

Because the potential energy stored in the organic substrate is released in small increments, it is easier to control oxidation and capture some of the energy as it is released—small energy transfers waste less energy than a single large transfer does.

2. Other Electron Acceptors

Most biological energy derives from oxidation of reduced metabolites in a series of reactions, with oxygen as the final electron acceptor.

Not all metabolic energy comes from oxidation by oxygen.

Substances other than oxygen can serve as terminal electron acceptors.

Many microorganisms either can or must live anaerobically.

For example, Desulfovibrio（脱硫弧菌） carry out anaerobic respiration using sulfate as the terminal electron acceptor:

Most anaerobic organisms, however, derive their energy from fermentations, which are energy-yielding catabolic pathways that proceed with no net change in the oxidation state of the products as compared with that of the substrates

Oxidation State 氧化态：
氧化态表示一个化合物中某个原子的氧化程度。形式氧化态是通过假设所有异核化学键都为100%离子键而算出来的。氧化态用阿拉伯数字表示，可以为正数、负数或是零。

3. Energy Yields, Respiratory Quotients, and Reducing Equivalents

Energy Yields

If metabolic energy comes primarily from oxidative reactions, it follows that the more highly reduced a substrate, the higher its potential for generating biological energy

The combustion of fat provides more heat energy than the combustion of an equivalent mass of carbohydrate.

In other words, fat has a higher caloric content（热焓, 含热量） than carbohydrate.

Compare the oxidation of glucose with the oxidation of a typical saturated fatty acid, palmitic acid（棕榈酸）.

Reducing Equivalents

Another way to express the degree of substrate oxidation is to say that more reducing equivalents（还原当量） are derived from oxidation of fat than from oxidation of carbohydrate.

We can also tell that glucose is the more highly oxidized substance because its oxidation produces more moles of per mole of consumed during oxidation, a ratio called the respiratory quotient, or RQ.

The above reaction stoichiometries reveal RQ for glucose to be 1.0(6CO2/6O2) whereas that for palmitic acid is 0.70(16CO2/23O2)

In general, the lower the RQ for a substrate, the more oxygen consumed per carbon oxidized and the greater the potential per mole of substrate for generating ATP.

A reducing equivalent can be defined as 1 mole of hydrogen atoms (one proton and one electron per H atom).

For example, two moles of reducing equivalents are used in the reduction of one-half mole of oxygen to water:

            ½ O2 + 2e- + 2H+ → H2O 

Whereas the breakdown of complex organic compounds yields both energy and reducing equivalents, the biosynthesis of such compounds utilizes both.

(1) NAD+/NADH And NADP+/NADPH

The major source of electrons for reductive biosynthesis is NADPH, nicotinamide adenine dinucleotide phosphate (reduced).

NADP+ and NADPH are identical to NAD+ and NADH, respectively, except that the former have an additional phosphate esterified at C-2 on the adenylate moiety.

NAD+ and NADP are equivalent in their thermodynamic tendency to accept electrons; they have equal standard reduction potentials (Chapter 3).

Nicotinamide nucleotides（烟酰胺核苷酸） in catabolism and biosynthesis:

NAD+ is the cofactor for most enzymes that act in the direction of substrate oxidation (dehydrogenases).

NADPH usually functions as a cofactor for reductases, enzymes that catalyze substrate reduction.

Nicotinamide nucleotide–linked enzymes that oxidize substrates (dehydrogenases) usually use and NAD+, those enzymes that reduce substrates (reductases) usually use NADPH

Of course, both dehydrogenases and reductases catalyze reversible reactions—the directions are determined by the redox state, or ratio of the oxidized and reduced forms of each pair that prevail in the cell.

Thus, the NAD+/NADH pair is maintained at a more oxidized level than the NADP+ /NADPH pair in a healthy cell. These ratios generally drive NAD+-linked reactions in the oxidative direction and NADP+-linked reactions in the reductive direction.

An exception is two -linked dehydrogenases in the pentose phosphate pathway (see Chapter 13), which convert to NADPH and represent a major route for regeneration of the reduced nucleotide.

ATP as a Free Energy Currency

ATP serves as an immediate donor of free energy—it is continuously formed and consumed.

There is no chemical necessity for the production of 32 moles of ATP (in fact, there is not even a direct chemical connection between glucose oxidation and ATP synthesis). We could not predict a stoichiometry of 32 ATP from chemical considerations.

This is instead an evolved-coupling stoichiometry. Evolved-coupling stoichiometries are biological adaptations, phenotypic traits acquired during evolution—they are the result of compromise

1. ATP-coupling coefficient

The fundamental biological role of ATP as an energy-coupling compound is to convert thermodynamically unfavorable processes into favorable processes.

The number of ATP (or ATP equivalents) produced in the catabolic direction will always be different from the number of ATP (or ATP equivalents) required in the anabolic direction

We refer to these numbers as the ATP-coupling coefficient of the reaction or pathway. For example, in the fructose-6-phosphate/fructose-1,6-bisphosphate substrate cycle (page 481), the glycolytic reaction,

has an ATP-coupling coefficient of The gluconeogenic reaction:

has an ATP-coupling coefficient of 0. Likewise, as we will learn in Chapter 13, glycolysis has an overall ATP-coupling coefficient of +2, whereas gluconeogenesis has an ATP-coupling coefficient of -6.

Coupling ATP hydrolysis to a pathway provides a new chemical route, using different reactions with different stoichiometries, resulting in a different overall equilibrium constant for the process.

(+)2. The Influence of Gradient of ATP/ADP

In fact, coupling ATP hydrolysis to a process changes the equilibrium ratio of certain [reactants] to [products] by a factor of 10^8^ !

To illustrate this, let’s consider how the equilibrium ratio of [fructose-1,6-bisphosphate] to [fructose-6-phosphate]:

In many cells, the normal intracellular concentration of Pi is ~ 1mM (10^-3^ M), so the equilibrium ratio of [fructose-1,6-bisphosphate] / [ fructose-6-phosphate] achieved by this reaction would be (0.0014)(10^-3^) = 1.4 X 10^-6^

Now let’s compare that to a reaction that couples the phosphorylation of fructose-6-phosphate to ATP hydrolysis, as in reaction 12.6 above. $\Delta G ^{\circ \prime}$ for this reaction is -14.2 kJ/mol (see Table 13.1, page 534), and thus the equilibrium constant, K, for this reaction is 308.

In most normal, healthy cells, [ATP] is several-fold (3–10 times) higher than [ADP].

For the purposes of this calculation, let’s make the simplifying assumption that the intracellular concentrations of ADP and ATP are roughly equal, that is, their concentration ratio is ~1.

Thus, [ADP] and [ATP] cancel out, and the equilibrium ratio of [fructose-1,6-bisphosphate]/[fructose-6-phosphate] for this reaction is 308.

This equilibrium ratio is indeed 10^8^ times higher than that achieved without ATP coupling [308/(1.4 X 10^-6^) = 2.2 X 10^8^].

3. Metabolite Concentrations and Solvent Capacity

Evolution has produced a complex cell metabolism comprising thousands of enzymes and metabolic intermediates each at very low individual concentrations.

There are at least a couple of reasons why a cell must maintain its components at very low concentrations:

1. Aqueous compartment has a finite capacity for the amount of dissolved substances (metabolites and macromolecules)—this is its solvent capacity. Thus, individual metabolites must exist at low concentrations (10^-3^-10^-6^M, or even lower) to avoid exceeding the solvent capacity of the cell
2. Low metabolite concentrations minimize unwanted side reactions.

ATP Coupling Help Avoid High Metabolite Concentrations

How does ATP coupling help avoid high metabolite concentrations? It does it by activating metabolic intermediates.

Activated intermediates, such as ATP, allow reactions to occur under physiologically relevant concentrations（有关生理浓度） of metabolic intermediates.

ATP: TWO phosphoanhydride bonds
ATP has a “high phosphoryl  group transfer potential”
ATP: “free energy currency”
Turn over fast (a resting human): 65 kg ATP/24 hrs!!!
Exercise: 0.5 kg ATP/min

1. Mechanical energy: muscle contract
2. Chemical energy
3. Osmotic energy: trasnporting againesy concentration gradient.
4. Electrical energy: nerve pulse

Cells have evolved a number of activated intermediates besides ATP, including acetyl-CoA, acyl-lipoate, and nucleoside diphosphate sugars (NDP-sugars)

Thermodynamic Properties of ATP

When we call ATP a “high-energy compound,” as we did in Chapter 3, we use that term within a defined context; i.e., a high-energy compound is one containing at least one bond with a sufficiently favorable $\Delta G ^{\circ \prime}$ of hydrolysis.

Note, however, that calling a substance a high-energy compound does not mean that it is chemically unstable or unusually reactive

ATP is a kinetically stable compound; its spontaneous hydrolysis is slow, but when hydrolysis does occur, whether spontaneously or enzyme-catalyzed, substantial free energy is released.

Phosphoanhydride（磷酸酐键） bonds are thermodynamically unstable, but kinetically stable—large free energies of activation require enzymes to lower the activation barrier.

It is more accurate to say that ATP has a “high phosphoryl group transfer potential” than to call it a high-energy compound

The electrostatic repulsion among the negative charges in the molecule before hydrolysis, resonance stabilization of the products of hydrolysis, and the tendency of the hydrolysis products to deprotonate

The above mentioned factors combine to give ATP hydrolysis a $\Delta$G°’ of -30.5 kJ/mol, twice the phosphate transfer potential of phosphate esters such as AMP.

On the other hand, several important metabolites have $\Delta $G°’ values for hydrolysis that are much more negative than that of ATP

This means that ATP is actually intermediate on the scale of “chemical potential.” This is important as well because it means that the breakdown of a compound such as phosphoenolpyruvate can be coupled to drive the synthesis of ATP itself from ADP and Pi.

In fact, such coupled reactions, called substrate-level phosphorylation reactions, represent the process by which ATP is synthesized in glycolysis, as we discuss in Chapter 13.

(+) 4. ATP is Chelated within Cells

Most ATP is chelated within cells, as a complex with Mg2+

ADP is also complexed with Mg2+ but ADP has a different affinity for Mg2+ from that of ATP. Varying levels of Mg2+ will change $\Delta G$ in complicated ways, depending upon relative affinities of reactants and products for the magnesium ion.

(+) The Important Differences Between $\Delta G$ And $\Delta G ^{\circ \prime}$

But what provides the energy for synthesis of compounds with a much higher phophate transfer potential than that of ATP itself? Much of the answer lies in the fact that $\Delta G$ values under intracellular conditions are quite different from standard ($\Delta G ^{\circ \prime}$) values.

This is mainly because intracellular concentrations are far different from the 1M concentrations used to compute standard free energies.

In fact, the physiological ratio of [ATP]/[ADP] is times higher than the equilibrium ratio.

As we shall see, maintenance of the physiological ratio of [ATP]/[ADP] so far away from equilibrium is accomplished by kinetic control, i.e., by regulation of enzymes. The key point is that maintaining the physiological ratio so far from equilibrium provides the thermodynamic driving force for nearly every biochemical event in the cell.

Kinetic Control of Substrate Cycles

ATP can drive the synthesis of higher-energy compounds, if nonequilibrium intracellular concentrations make such reactions exergonic.

Enzymes in substrate cycles are kinetically regulated by the levels of allosteric effectors.

The choice of which pathway operates, however, is determined entirely by the metabolic needs of the cell (via allosteric effectors), not by thermodynamics (because both pathways are favorable).

Why is it so important for both pathways to be thermodynamically favorable at all times? Because regulation can be imposed only on reactions that are displaced far from equilibrium. Consider the following analogy:

A dam separates two bodies of water. If the water level is the same on both sides of the dam, we can say the system is at equilibrium. If we now open the floodgate, what happens? Water may move back and forth through the floodgate, but there will be no net movement of water or change in water levels. Thus, regulation (opening the floodgate) has no impact on a system at equilibrium. Now imagine a different system.

Other High-Energy Phosphate Compounds

This same principle allows ATP to drive the synthesis of compounds of even higher phosphate transfer potential, such as creatine phosphate

Creatine phosphate (CrP) is produced from creatine by the enzyme creatine kinase:

Cr: creatine （肌氨酸,肌肉素）
CrP: creatine phosphate（[化]磷酸肌酸）
CK: creatine kinase（肌酸激酶）

Mammalian cells have several different creatine kinase isozymes, one of which is localized to the intermembrane space of mitochondria (mCK; see margin).

However, because ATP levels are high within mitochondria, and creatine phosphate levels are relatively low the reaction is exergonic as written and proceeds to the right in the mitochondrial intermembrane space.

ATP can also drive the synthesis of compounds of even higher phosphate transfer potential, such as creatine phosphate.

This compound shuttles phosphate bond energy from ATP in mitochondria to myofibrils, where that bond energy is transduced to the mechanical energy of muscle contraction.

Creatine phosphate (CrP) is produced from creatine by the enzyme creatine kinase:

In some invertebrate animals arginine phosphate, instead of creatine phosphate, plays a similar role in storing high-energy phosphate for rapid production, as needed, of ATP.

(+) Other High-Energy Nucleotides

Evolution has created an array of enzymes that preferentially bind ATP and use its chemical potential to drive endergonic reactions

In most cells, ATP levels, at 2–8 mM, are several-fold higher than those of the other nucleoside triphosphates and also several-fold higher than the levels of ADP or AMP. These factors give ATP a strong tendency to distribute its $\gamma$ (outermost) phosphate in the synthesis of other nucleoside triphosphates.

This is accomplished through the action of nucleoside diphosphate kinase, which synthesizes CTP from CDP in the following example.

Some metabolic reactions, such as the activation of amino acids for protein synthesis, cleave ATP, not to ADP and Pi but to AMP and PPi

The conversion of AMP to ATP, allowing reuse of the nucleotide, involves another enzyme, adenylate kinase (also called myokinase because of its abundance in muscle).

五、Major Metabolic Control Mechanisms

Enzyme levels: expression and degradation.

Enzyme activity is regulated by interaction with substrates, products, and allosteric effectors and by covalent modification of enzyme protein.

Compartmentation

Hormonal regulation （glucagon， cAMP）

Distributive control of metabolism（代谢的分配控制）.

(+) Control of Enzyme Levels

Through synthesis of new protein, from less than one molecule per cell to many thousands of molecules per cell.

This phenomenon is called enzyme induction. Similarly, the presence of the end product of a pathway may turn off the synthesis of enzymes needed to generate that end product, a process called repression.

(+) Control of Enzyme Activity

The catalytic activity of an enzyme molecule can be controlled in two ways: by reversible interaction with ligands (substrates, products, or allosteric modifiers) and by covalent modification of the protein molecule

Enzyme activity is most commonly controlled by low-molecular-weight ligands, principally substrates and allosteric effectors.

For example, protein–protein interactions can affect enzyme activity, and several enzymes of nucleic acid metabolism are activated by binding to DNA.

Chapter 11 introduced several types of covalent modification that are used to regulate enzyme activity, including phosphorylation, acetylation, methylation, adenylylation (the transfer of an adenylate moiety from ATP), and ADP-ribosylation (the transfer of an ADP-ribosyl moiety from NAD+).

Many other less common covalent modifications are now known.

However, phosphorylation is by far the most widespread covalent modification used to control enzyme activity.

Control through covalent modification is often associated with regulatory cascades. Modification activates an enzyme, which in turn acts on a second enzyme, which may activate yet a third enzyme, which finally acts on the substrate.

Because enzymes act catalytically, this cascading provides an efficient way to amplify the original biological signal.

Compartmentation

Compartmentation creates a division of labor within a cell, which increases the efficiency of cell function. The creatine–creatine phosphate shuttle (page 496) is a good example of this.

1. Physical Compartmentation

Locations of major metabolic pathways within a eukaryotic cell:
This hypothetical cell combines features of a plant cell and an animal cell.

Typically, intermediates of a pathway remain trapped within an organelle, while specific carriers allow substrates to enter and products to exit. The flux through a pathway, therefore, can be regulated by controlling the rate at which a substrate enters the compartment.

Juxtaposition (并列) of enzymes that catalyze sequential reactions localizes substrates even in the absence of membrane-bound organelles.

(+) 2. Weak Interactions Caused Compartmentation

Compartmentation can also result from weak interactions among enzymes that do not remain complexed when they are isolated.

For example, conversion of glucose to pyruvate by glycolysis is catalyzed by enzymes that interact quite weakly in solution. However, there is evidence that these enzymes interact within the cytosol, forming a supramolecular structure that facilitates the multistep glycolytic pathway.

The cytosol is much more highly structured than was formerly thought.

It has been proposed that soluble enzymes are bound within the cell to the structural elements of the cytomatrix

Whether highly structured or loosely associated, multienzyme complexes allow for efficient control of reaction pathways. Enzyme complexes restrict diffusion of intermediates, thereby keeping the average concentrations of intermediates low (but their local concentrations high, at enzyme catalytic sites). T

Hormonal regulation

Second messengers（第二信使） transmit information from hormones bound at the cell surface, thereby controlling intracellular metabolic processes.

第二信使（英语：Second messenger）在生物学里是胞内信号分子，负责细胞内的信号转导以触发生理变化，如增殖，细胞分化，迁移，存活和细胞凋亡。因此第二信使是细胞内的信号转导的启动组成部件之一。第二信使分子的例子包括：环腺苷酸（cAMP），环磷酸鸟苷（cGMP），肌醇三磷酸（IP3），甘油二酯（DAG），钙离子（Ca）。细胞释放第二信使分子是响应于暴露在细胞外的信号分子-第一信使。第一信使是细胞外因子，通常是激素或神经递质，如肾上腺素，生长激素，和血清素。

The process of transmitting these messages and bringing about metabolic changes is called signal transduction. The extracellular messengers include hormones, growth factors, neurotransmitters, and pheromones, which interact with specific receptors, resulting in specific metabolic changes in the target cell.

Metabolic responses to hormones can involve changes in gene expression, leading to changes in enzyme levels. This type of response typically operates on a timescale of hours to days, resulting in a reprogramming of the metabolic capability of the cell.

On a shorter timescale (seconds to hours), some hormones stimulate the synthesis of intracellular second messengers that control metabolic reactions.

One of the most important second messengers is adenosine 3’,5’-cyclic monophosphate, more commonly called cyclic AMP, or simply cAMP.

(+) 1. Modular Signal Transduction Systems

Signal transduction systems are modular in nature, allowing for a diversity of metabolic responses based on the same operating principles. Thus, secretion of one hormone can have quite diverse effects in different tissues, depending upon the nature of the receptor and other components and second messenger systems in different target cells. Moreover, a single second messenger may have diverse effects within a single cell.

(+) Distributive Control of Metabolism

We now realize that metabolic regulation is more complex and that all of the enzymes in a pathway contribute toward control of pathway flux.

An approach called metabolic control analysis assigns to each enzyme in a pathway a flux control coefficient, $C^{J}$, a value that can vary between zero and one.

The flux through the pathway, J, is equal to the rate of the forward process, minus

the rate of the reverse process, $v_{r}$:
$$
J = v_{f} - v_{r}
$$
For a given enzyme, the flux control coefficient is the relative increase in flux, divided by the relative increase in enzyme activity that brought about that flux increase.

For a true rate-limiting enzyme, the flux control coefficient is 1; a 20% increase in the activity of that enzyme would increase that flux rate by 20%.

Flux-control coefficients are properties of the pathway or metabolic system, and thus all the flux-control coefficients in the pathway must sum to 1:
$$
C_{1}^{J} + C_{2}^{J} + C_{3}^{J} + ··· + C_{n}^{J} = 1
$$
The predictions of metabolic control theory can be tested, for example, by using mutations affecting a specific enzyme to bring about defined changes in the activity of that enzyme in vivo

every enzyme in a pathway contributes toward control of that pathway; in other words, every enzyme has a flux-control coefficient greater than zero.

Thus, regulation of a pathway is distributed among all of the enzymes involved in the pathway, giving rise to the concept of distributive control of metabolism.

Levels of Organization at Which Metabolism Is Studied

1. Whole organism: Radioactive tracers; 14C-acetate-cholesterol synthesis; GTT; NMR,; noninvasive MRI.
2. Isolated or perfused organs.
3. Whole cells: trypsin, collagenase, cellulase, FACS sorting, tissue, cell line.
4. Cell-free system: shear forces, hypotonic media, freezing and thawing, sonication, lysozyme, homogenation.
5. Purified compartments:
6. Purified proteins: citric acid cycle, reconstitution

六、Experimental Analysis of Metabolism

NMR（核磁共振）

Example: Living organisms: ^31^P-NMR spectra

Effect of anaerobic exercise on 31P-NMR spectra of human forearm muscle:

1. Before exercise.

2. One minute into a 19-minute exercise period.

3. The 19^th^ minute.

4. Ten minutes after exercise.

Peak areas are proportional to intracellular concentrations.

Peak 1 = Pi; 2 = creatine phosphate; 3 = ATP γ-phosphate; 4 = ATP α-phosphate; 5 = ATP β-phosphate; 6 = phosphomonoesters.

NMR: nuclear magnetic resonance 核磁共振

Metabolic Probes

CO, cyanide(氰化物): respiratory chain studies

By inactivating individual enzymes, mutations and enzyme inhibitors help identify the metabolic roles of enzymes.

The steps of a hypothetical metabolic pathway are identified by analysis of mutants defective in individual steps of the pathway.

We can identify metabolite C as the substrate for enzyme III by the absence of this enzyme in mutants that accumulate C.

We know that D and E follow C in the pathway because feeding either D or E to mutants defective in enzyme III bypasses the genetic block and allows the cells to grow.

七、Radioisotopes and the Liquid Scintillation Counter

Radioisotopes revolutionized biochemistry when they became available to investigators shortly after World War II.

Radioisotopes extend by orders of magnitude the sensitivity with which chemical species can be detected.

Traditional chemical analysis can detect and quantify molecules in the micromole or nanomole range (i.e., 10-6 to 10-9 mole).

A compound that is “labeled,” containing one or more atoms of a radioisotope, can be detected in picomole or even femtomole amounts (i.e., 10-12 to 10-15 mole).

Radiolabeled compounds are called tracers because they allow an investigator to follow specific chemical or biochemical transformations in the presence of a huge excess of nonradioactive material.

Of the many uses of stable isotopes in biochemical research, we mention three applications here.

First, incorporation of a stable isotope often increases the density of a material because the rare isotopes usually have higher atomic weights than their more abundant counterparts.

• This difference presents a way to separate labeled from nonlabeled compounds physically, as in the Meselson–Stahl experiment on DNA replication.

Second, compounds labeled with stable isotopes, particularly are widely used in NMR studies of molecular structure and dynamics.

Third, stable isotopes are used to study reaction mechanisms. The “kinetic isotope effect” refers to the effect on reaction rate of substitution of an atom by a heavy isotope.

八、Metabolomics

Metabolomics is the scientific study of chemical processes involving metabolites. Specifically, metabolomics is the “systematic study of the unique chemical fingerprints that specific cellular processes leave behind”, the study of their small-molecule metabolite profiles.

（代谢物组学（英语：metabolomics）是在后基因组学时代兴起的一门跨领域学科，其主要目标是定量的研究生命体对外界刺激、病理生理变化、以及本身基因突变而产生的其体内代谢物水平的多元动态反应。）

The development of these new technologies has driven the “-omics” revolution:

• Genomics
• Transcriptomics
• Proteomics
• Metabolomics

Hundreds or even thousands of specific components are measured simultaneously in a biological sample.

Thus, it is now feasible to measure the full set of transcripts (transcriptome 转录组), proteins (proteome（蛋白质组）), or metabolites (metabolome（代谢组）) in a particular cell or tissue.

The metabolome represents the ultimate molecular phenotype of a cell under a given set of conditions because all the changes in gene expression and enzyme activity eventually lead to changes in cellular metabolite levels (the metabolic state or profile).

Basic Process

Basic process of metabolic profiling:

Metabolites are identified and quantified by an analytical method.

Data are collected and visualized by informatics approaches.

Informatics approaches are then used to reveal relationships and patterns among the samples.

Differential metabolic profile from livers of control mice and livers from mice 2 h after treatment with acetaminophen（对乙酰氨基酚）:

The 2D plot (m/z vs. elution time) shows cations detected by CE-MS analysis.

The arrow points to the metabolite subsequently identified as ophthalmate（邻苯二甲酸酯） whose level significantly increased in treated mice.

The color bar indicates the increase (red) or the decrease (blue) of metabolite level after drug treatment.

Biomarkers

Example: Diabetic Studies

Example: CML Patients

Principal component analysis of blood plasma metabolites in chronic myeloid leukemia (CML) patients:

3D scatter plot of the first three principal components (PC1 vs. PC2 vs. PC3).

Green squares are patients who are sensitive to drug treatment (SCML).

Black diamonds are patients who are resistant to drug treatment (RCML).

Red triangles are untreated CML patients (UCML).

Blue circles are healthy controls (HC).