Biochemistry, 4th Edition P90 pptx

These hormones act in the brain, principally Glycogen Glucose-6-phosphate Pyruvate Glycolysis Acetyl-CoA Oxidative phosphorylation Citric acid cycle CO2 + H2O Cholesterol Fatty acids Fat

brain, and other tissues If energy demands are low, fatty acids are incorporated into

triacylglycerols that are carried to adipose tissue for deposition as fat Cholesterol is

also synthesized in the liver from two-carbon units derived from acetyl-CoA.

In addition to these central functions in carbohydrate and fat-based energy

me-tabolism, the liver serves other purposes For example, the liver can use amino acids

as metabolic fuels Amino acids are first converted to their corresponding -keto acids

by aminotransferases The amino group is excreted after incorporation into urea in

the urea cycle The carbon skeletons of glucogenic amino acids can be used for

glu-cose synthesis, whereas those of ketogenic amino acids appear in ketone bodies (see

Figure 25.41) The liver is also the principal detoxification organ in the body The

endoplasmic reticulum of liver cells is rich in enzymes that convert biologically active

substances such as hormones, poisons, and drugs into less harmful by-products

Liver disease leads to serious metabolic derangements, particularly in amino acid

metabolism In cirrhosis, the liver becomes defective in converting NH4 to urea for

excretion, and blood levels of NH4 rise Ammonia is toxic to the central nervous

system, and coma ensues.

27.6 What Regulates Our Eating Behavior?

Approximately 65% of Americans are overweight, and one in three Americans is

clin-ically obese (overweight by 20% or more) Obesity is the single most important cause

of type 2 (adult-onset insulin-independent) diabetes Research into the regulatory

controls that govern our feeding behavior has become a medical urgency with great

financial incentives, given the epidemic proportions of obesity and widespread

pre-occupation with dieting and weight loss

The Hormones That Control Eating Behavior Come From

Many Different Tissues

Appetite and weight regulation are determined by a complex neuroendocrine

sys-tem that involves hormones produced in the stomach, small intestines, pancreas,

adi-pose tissue, and central nervous system These hormones act in the brain, principally

Glycogen

Glucose-6-phosphate

Pyruvate Glycolysis

Acetyl-CoA

Oxidative phosphorylation

Citric acid cycle

CO2 + H2O

Cholesterol Fatty acids

Fatty acid synthesis

Triacylglycerols,

phospholipids

Lipid

synthesis

Blood glucose

and pentose phosphates

Pentose phosphate pathway

ATP

ATP

NADPH

FIGURE 27.11 Metabolic conversions of glucose-6-phosphate in the liver

854 Chapter 27 Metabolic Integration and Organ Specialization

on neurons within the arcuate nucleus region of the hypothalamus The arcuate nu-cleus is an anatomically distinct brain area that functions in homeostasis of body weight, body temperature, blood pressure, and other vital functions (Figure 27.12) The neurons respond to these signals by activating, or not, pathways involved in eat-ing (food intake) and energy expenditure Hormones that regulate eateat-ing behavior can be divided into short-term regulators that determine individual meals and long-term regulators that act to stabilize the levels of body fat deposits Two subsets of

neurons are involved: (1) the NPY/AgRP-producing neurons that release NPY

(neuropeptide Y), the protein that stimulates the neurons that trigger eating

behav-ior, and (2) the melanocortin-producing neurons, whose products inhibit the

neu-rons initiating eating behavior AgRP is agouti-related peptide, a protein that blocks

the activity of melanocortin-producing neurons Melanocortins are a group of peptide

hormones that includes ␣- and ␤-melanocyte–stimulating hormones (␣-MSH and

␤-MSH) Melanocortins act on melanocortin receptors (MCRs), which are members

of the 7-TMS G-protein–coupled receptor (GPCR) family of membrane receptors; MCRs trigger cellular responses through adenylyl cyclase activation (see Chapters 15 and 32).

Ghrelin and Cholecystokinin Are Short-Term Regulators

of Eating Behavior Short-term regulators of eating include ghrelin and cholecystokinin Ghrelin is an

appetite-stimulating peptide hormone produced in the stomach Production of ghrelin is maximal when the stomach is empty, but ghrelin levels fall quickly once food is consumed Cholecystokinin is a peptide hormone released from the

gas-Insulin

Pancreas Fat tissue

producing neuron

Hypothalamus

Food intake

Energy expenditure Neuron

NPY/

Melanocortin receptor (MC4R) (blocked by AgRP) Ghrelin receptor NPY/PYY3-36 receptor Y2R Melanocortin receptor (MC3R)

Leptin receptor

or insulin receptor

NPY receptor Y1R Stomach

Leptin

Ghrelin PYY3-36

ⴙ

ⴙ ⴚ

ⴚ

ⴚ

Intestines

FIGURE 27.12 The regulatory pathways that control eating.(Adapted from Figure 1 in Schwartz, M W., and Morton,

trointestinal tract during eating In contrast to ghrelin, cholecystokinin signals

sati-ety (the sense of fullness) and tends to curtail further eating Together, ghrelin and

cholecystokinin constitute a meal-to-meal control system that regulates the onset

and end of eating behavior The activity of this control system is also modulated by

the long-term regulators.

Insulin and Leptin Are Long-Term Regulators of Eating Behavior

Long-term regulators include insulin and leptin, both of which inhibit eating and

promote energy expenditure Insulin is produced in the -cells of the pancreas

when blood glucose levels rise A major role of insulin is to stimulate glucose uptake

from the blood into muscle, fat, and other tissues Blood insulin levels correlate with

body fat amounts Insulin also stimulates fat cells to make leptin Leptin (from the

Greek word lepto, meaning “thin”) is a 16-kD, 146–amino acid residue protein

pro-duced principally in adipocytes (fat cells) Leptin has a four-helix bundle tertiary

structure similar to that of cytokines (protein hormones involved in cell–cell

com-munication) Normally, as fat deposits accumulate in adipocytes, more and more

lep-tin is produced in these cells and spewed into the bloodstream Leplep-tin levels in the

blood communicate the status of triacylglycerol levels in the adipocytes to the

cen-tral nervous system so that appropriate changes in appetite take place If leptin

lev-els are low (“starvation”), appetite increases; if leptin levlev-els are high (“overfeeding”),

appetite is suppressed Leptin also regulates fat metabolism in adipocytes, inhibiting

fatty acid biosynthesis and stimulating fat metabolism In the latter case, leptin

in-duces synthesis of the enzymes in the fatty acid oxidation pathway and increases

ex-pression of uncoupling protein 2 (UCP2), a mitochondrial protein that uncouples

oxi-dation from phosphorylation so that the energy of oxioxi-dation is lost as heat

(thermogenesis) Leptin binding to leptin receptors in the hypothalamus inhibits

re-lease of NPY Because NPY is a potent orexic (appetite-stimulating) peptide hormone,

leptin is therefore an anorexic (appetite-suppressing) agent Functional leptin

recep-tors are also essential for pituitary function, growth hormone secretion, and normal

puberty When body fat stores decline, the circulating levels of leptin and insulin also

decline Hypothalamic neurons sense this decline and act to increase appetite to

re-store body fat levels.

Intermediate regulation of eating behavior is accomplished by the gut hormone

PYY3-36 PYY3-36is produced in endocrine cells found in distal regions of the small

in-testine, areas that receive ingested food some time after a meal is eaten PYY3-36

inhibits eating for many hours after a meal by acting on the NPY/AgRP-producing

neurons in the arcuate nucleus Clearly, the regulatory controls that govern eating

are complex and layered Some believe that defects in these controls are common

and biased in favor of overeating, an advantageous evolutionary strategy that may

have unforeseen consequences in these bountiful times.

HUMAN BIOCHEMISTRY

The Metabolic Effects of Alcohol Consumption

Ethanol metabolism alters the NAD/NADH ratio Ethanol is

metabolized to acetate in the liver by alcohol dehydrogenase and

aldehyde dehydrogenase:

CH3CH2OH NAD34 CH3CHO NADH  H

CH3CHO NAD H2O34 CH3COO NADH  2H

Acetate is then converted to acetyl-CoA Excessive conversion of

available NADto NADH impairs NAD-requiring reactions, such

as the citric acid cycle, gluconeogenesis, and fatty acid oxidation

Accumulation of acetyl-CoA favors fatty acid synthesis, which,

along with blockage of fatty acid oxidation, causes elevated

triacyl-glycerol levels in the liver Over time, these triacyltriacyl-glycerols accu-mulate as fatty deposits, which ultimately contribute to cirrhosis of the liver Impairment of gluconeogenesis leads to buildup of this pathway’s substrate, lactate Lactic acid accumulation in the blood causes acidosis A further consequence is that acetaldehyde can form adducts with protein ONH2groups, which may inhibit pro-tein function Because gluconeogenesis is limited, alcohol

con-sumption can cause hypoglycemia (low blood sugar) in someone

who is undernourished In turn, hypoglycemia can cause irre-versible damage to the central nervous system

856 Chapter 27 Metabolic Integration and Organ Specialization

AMPK Mediates Many of the Hypothalamic Responses

to These Hormones

The actions of leptin, ghrelin, and NPY converge at AMPK Leptin inhibits AMPK activity in the arcuate nucleus, and this inhibition underlies the anorexic effects of leptin Leptin action on AMPK depends on a particular melanocortin receptor type

known as the melanocortin-4 receptor (MC4R) On the other hand, ghrelin and

NPY activate hypothalamic AMPK, which stimulates food intake and leads, over time, to increased body weight The effects of AMPK in the hypothalamus that lead

to alterations in eating behavior may be mediated through changes in malonyl-CoA levels Low [malonyl-CoA] in hypothalamic neurons is associated with increased food intake, and elevated malonyl-CoA levels are associated with suppression of eat-ing The inhibition of acetyl-CoA carboxylase (and thus, malonyl-CoA synthesis) as

a result of phosphorylation by AMPK plays an important part in the regulation of our eating behavior

27.7 Can You Really Live Longer by Eating Less?

Caloric Restriction Leads to Longevity

Nutritional studies published in 1935 showed that rats fed a low-calorie, but balanced and nutritious diet lived nearly twice as long as rats with unlimited access to food (2.4 years versus 1.3 years) Subsequent research over the ensuing 70 years has shown that the relationship between diet and longevity is a general one for organisms from

yeast to mammals To achieve this effect of caloric restriction (CR), animals are given

a level of food that amounts to 60% to 70% of what they would eat if they were al-lowed free access to food In animals, CR results in lower blood glucose levels, de-clines in glycogen and fat stores, enhanced responsiveness to insulin, lower body temperature, and diminished reproductive capacity The extended life span given by

CR offers a definite evolutionary advantage: Any animal that could slow the aging process and postpone reproduction in times of food scarcity and then resume re-production when food became available would out-compete animals without such ability.

Another remarkable feature of CR is that it diminishes the likelihood for develop-ment of many age-related diseases, such as cancer, diabetes, and atherosclerosis Is this benefit simply the result of lowered caloric intake, or does CR lead to significant reg-ulatory changes that affect many aspects of an organism’s physiology? The answer to this and other questions is emerging from vigorous research efforts toward under-standing CR.

Mutations in the SIR2 Gene Decrease Life Span

Many important clues came from genetic investigations Deletion of a gene termed

SIR2 (for silent information regulator 2) abolished the ability of CR to lengthen life

span in yeast and roundworms, implicating the SIR2 gene product in longevity SIR2

originally was discovered through its ability to silence the transcription of genes that

encode rRNA SIR2-related genes are found in some prokaryotes and virtually all

eukaryotes, including yeast, nematodes, fruit flies, and humans The human gene is

designated SIRT1, for sirtuin 1; sirtuin is the generic name for proteins encoded by

SIR2 genes Sirtuins are NAD-dependent protein deacetylases (Figure 27.13) Cleav-age of acetyl groups from proteins is an exergonic reaction, as is cleavCleav-age of the

N-glycosidic bond in NAD( G°  34 kJ/mol) Thus, involvement of NADin the reaction is not a thermodynamic necessity However, NAD+ involvement does couple the reaction to an important signal of metabolic status, namely, the NAD/NADH ratio Furthermore, both nicotinamide and NADH are potent in-hibitors of the deacelylase reaction Thus, the NAD/NADH ratio controls sirtuin protein deacetylase activity, so oxidative metabolism, which drives conversion of

NADH to NAD, enhances sirtuin activity One adaptive response found with CR is

increased mitochondrial biogenesis in liver, fat, and muscle, which is a response that

would raise the NAD/NADH ratio.

Sirtuin-catalyzed removal of acetyl groups from lysine residues of histones, the

core proteins of nucleosomes, allows the nucleosomes to interact more strongly

with DNA, making transcription more difficult (see Chapter 29 for a discussion of

the relationship between histone acetylation and transcriptional activity of genes).

SIRT1 Is a Key Regulator in Caloric Restriction

As a key regulator in CR, the human sirtuin protein SIRT1 connects nutrient

avail-ability to the expression of metabolic genes A striking feature of CR is the loss of fat

stores and reduction in white adipose tissue (WAT) SIRT1 participates in the

tran-scriptional regulation of adipogenesis through interaction with PPAR ␥ (peroxisome

proliferator-activator receptor- ), a nuclear hormone receptor that activates

tran-scription of genes involved in adipogenesis and fat storage SIRT1 binding to PPAR 

represses transcription of these genes, leading to loss of fat stores Because adipose

tissue functions as an endocrine organ, this loss of fat has significant hormonal

con-sequences for energy metabolism.

In liver, SIRT1 interacts with and deacetylates PGC-1 (peroxisome

proliferator-activator receptor-  coactivator-1), a transcriptional regulator of genes involved in

glucose production Thus, CR leads to increased transcription of the genes

encod-ing the enzymes of gluconeogenesis and repression of genes encodencod-ing glycolytic

en-zymes Acting in these roles, SIRT1 connects nutrient availability to the regulation

of major pathways of energy storage (glycogen and fat) and fuel utilization.

Resveratrol, a Compound Found in Red Wine, Is a Potent Activator

of Sirtuin Activity

Resveratrol (trans-3,4,5-trihydroxystilbene, Figure 27.14) is a phytoalexin

Phyto-alexins are compounds produced by plants in response to stress, injury, or fungal

infection Resveratrol is abundant in wine grape skins as a result of common

envi-ronmental stresses, such as infection by Botrytis cinerea, a fungus important in

mak-ing certain wines Because the skins are retained when grapes are processed to

+

+ +

O

HO OH

H2C

NH2

O

C O

N+

NH2 C O

N+

O

H3C

C

NH

Peptide

Acetyl-peptide

NAD +

Deacylated-peptide Nicotinamide

NH2

O OH

O

HO

2

-O-acetyl-ADP-ribose

OH

H2C

O–

O

NH2 N N N N

O

CH3 C

O

O–

O

O–

O

O–

O

O

O

HO OH

CH2

CH2

NH2 N N N N

FIGURE 27.13 The NAD-dependent protein deacetyl-ase reaction of sirtuins Acetylated peptides include

N-acetyl lysine side chains of histone H3 and H4 and

acetylated p53 (p53 is the protein product of the p53

tumor suppressor gene)

FIGURE 27.14 Resveratrol, a phytoalexin, is a member of the polyphenol class of natural products As a polyphe-nol, resveratrol is a good free-radical scavenger, which may account for its cancer preventative properties

OH

OH HO

Resveratrol (trans-3,4ⴕ,5-trihydroxystilbene)

858 Chapter 27 Metabolic Integration and Organ Specialization

make red wines, red wine is an excellent source of resveratrol Resveratrol might be the basis of the French paradox—the fact that the French people enjoy longevity and relative freedom from heart disease despite a high-fat diet When resveratrol is

given to yeast cultures, roundworms (Caenorhabditis elegans), or fruit flies (Drosophila melanogaster), it has the same life-extending effects as CR Resveratrol increased the

replicative life span (the number of times a cell can divide before dying) of yeast by 70% Resveratrol activates SIRT1 NAD-dependent deacetylase activity Further-more, resveratrol activates AMPK in the brain and in cultured neurons Because AMPK is a key energy sensor, resveratrol’s influences on longevity may arise through its effects on caloric homeostasis.

Because the effects of CR and resveratrol on longevity are not additive, a reason-able conclusion is that they operate via a common mechanism If this is so, then if you want to enjoy longevity, the appropriate advice would be “Eat less or drink red wine,” not “Eat less and drink red wine”!

SUMMARY

27.1 Can Systems Analysis Simplify the Complexity of Metabolism?

Cells are in a dynamic steady state maintained by considerable metabolic

flux The metabolism going on in even a single cell is so complex that it

defies meaningful quantitative description Nevertheless, overall

relation-ships become more obvious by a systems analysis approach to

intermedi-ary metabolism The metabolism of a typical heterotrophic cell can be

summarized in three interconnected functional blocks: (1) catabolism,

(2) anabolism, and (3) macromolecular synthesis and growth

Photo-trophic cells require a fourth block: photosynthesis Only a few metabolic

intermediates connect these systems, and ATP and NADPH serve as the

carriers of chemical energy and reducing power, respectively, between

these various blocks

27.2 What Underlying Principle Relates ATP Coupling to the

Thermo-dynamics of Metabolism? ATP coupling determines the

thermody-namics of metabolic sequences The ATP coupling stoichiometry

can-not be predicted from chemical considerations; instead it is a quantity

selected by evolution and the fundamental need for metabolic

se-quences to be emphatically favorable from a thermodynamic

perspec-tive Catabolic sequences generate ATP with an overall favorable Keq

(and hence, a negative G) and anabolic sequences consume this

en-ergy with an overall favorable Keq, even though such sequences may

span the same starting and end points (as in fatty acid oxidation of

palmitoyl-CoA to 8 acetyl-CoA versus synthesis of palmitoyl-CoA from

8 acetyl-CoA) ATP has two metabolic roles: a stoichiometric role in

rendering metabolic sequences thermodynamically favorable and a

reg-ulatory role as an allosteric effector

27.3 Is There a Good Index of Cellular Energy Status? The level of

phosphoric anhydride bonds in the adenylate system of ATP, ADP, and

AMP can be expressed in terms of the energy charge equation:

Energy charge 1

More revealing of the potential for an ATP-dependent reaction to occur

is, the phosphorylation potential:   [ATP]/([ADP][Pi]

27.4 How Is Overall Energy Balance Regulated in Cells? AMP-activated

protein kinase (AMPK) is the cellular energy sensor When cellular

energy levels are high, as signaled by high ATP concentrations, AMPK

is inactive When cellular energy levels are depleted, as signaled by

high [AMP], AMPK is allosterically activated and phosphorylates many

enzymes involved in cellular energy production and consumption

Competition between AMP and ATP for binding to the AMPK allosteric

sites determines AMPK activity AMPK activation leads to elevated

energy production and diminished energy consumption AMPK is an

-heterotrimer The -subunit is the catalytic subunit AMP or ATP

2 [ATP]  [ADP]

[ATP]  [ADP]  [AMP]

binding to the -subunit determines AMPK activity, with AMP binding

increasing activity by more than 1000-fold

Activation of AMPK leads to phosphorylation of key enzymes in en-ergy metabolism, activating those involved in ATP production and inac-tivating those in ATP consumption In addition, AMPK phosphorylation

of various transcription factors leads to elevated expression of catabolic genes and diminished expression of genes encoding biosynthetic zymes Beyond these cellular effects, AMPK plays a central role in en-ergy balance in multicellular organisms, because its activity is responsive

to hormones that govern eating behavior and energy homeostasis

27.5 How Is Metabolism Integrated in a Multicellular Organism? Organ systems in complex multicellular organisms carry out specific physiologi-cal functions, with each expressing those metabolic pathways appropriate

to its physiological purpose Essentially all cells in animals carry out the central pathways of intermediary metabolism, especially the reactions of ATP synthesis Nevertheless, organs differ in the metabolic fuels they pre-fer as substrates for energy production The major fuel depots in animals are glycogen in liver and muscle; triacylglycerols (fats) in adipose tissue; and protein, most of which is in skeletal muscle The order of preference for the use of these fuels is glycogen triacylglycerol protein Never-theless, the tissues of the body work together to maintain caloric home-ostasis: Constant availability of fuels in the blood The major organ systems have specialized metabolic roles within the organism

The brain has a strong reliance on glucose as fuel Muscle at rest pri-marily relies on fatty acids, but under conditions of strenuous contrac-tion when O2is limiting, muscle shifts to glycogen as its primary fuel The heart is a completely aerobic organ, rich in mitochondria, with a preference for fatty acids as fuel under normal operating conditions The liver is the body’s metabolic processing center, taking in nutrients and sending out products such as glucose, fatty acids, and ketone bod-ies Adipose tissue takes up glucose and, to a lesser extent, fatty acids for the synthesis and storage of triacylglycerols

27.6 What Regulates Our Eating Behavior? Appetite and weight reg-ulation are governed by hormones produced in the stomach, small in-testines, pancreas, adipose tissue, and central nervous system These hormones act on neurons within the arcuate nucleus region of the hy-pothalamus that control pathways involved in eating (food intake) and energy expenditure Hormones that regulate eating behavior can be di-vided into short-term regulators that determine individual meals and long-term regulators that act to stabilize the levels of body fat deposits Short-term regulators of eating include ghrelin and cholecystokinin Ghrelin is produced when the stomach is empty, but ghrelin levels fall quickly once food is consumed Cholecystokinin is released from the gastrointestinal tract during eating In contrast to ghrelin,

chole-cysokinin signals satiety (the sense of fullness) and tends to curtail

fur-ther eating Togefur-ther, ghrelin and cholecystokinin constitute a

meal-to-meal control system that regulates the onset and end of eating behavior

Long-term regulators include insulin and leptin, both of which

in-hibit eating and promote energy expenditure Blood insulin levels

cor-relate with body fat amounts Insulin also stimulates fat cells to make

leptin Leptin is produced principally in adipocytes As fat accumulates

in adipocytes, more leptin is released into the bloodstream to

commu-nicate the status of adipocyte fat to the central nervous system If leptin

levels are low (“starvation”), appetite increases; if leptin levels are high

(“overfeeding”), appetite is suppressed Leptin binding to its receptors

in the hypothalamus inhibits release of NPY NPY is a potent orexic

hor-mone; therefore, leptin is an anorexic agent When body fat stores

de-cline, the circulating levels of leptin and insulin also decline

Hypothal-amic neurons sense this decline and act to increase appetite to restore

body fat levels

Intermediate regulation of eating behavior is accomplished by the

gut hormone PYY336 Produced in distal regions of the intestines,

PYY336delays eating for many hours after a meal by inhibiting the

NPY/AgRP-producing neurons in the arcuate nucleus The regulatory

controls that govern eating are complex and layered

27.7 Can You Really Live Longer by Eating Less? Caloric restriction

(CR) prolongs the longevity of organisms from yeast to mammals CR

results in lower blood glucose levels, decline in glycogen and fat stores,

enhanced responsiveness to insulin, lower body temperature, and

di-minished reproductive capacity CR also diminishes the likelihood for

development of many age-related diseases, such as cancer, diabetes, and

atherosclerosis Genetic investigations revealed that mutations in the SIR2 gene abolish the extension of life span by CR The human SIR2 gene equivalent is SIRT1 SIRT genes encode sirtuins, a family of NAD -dependent protein deacetylases The NAD/NADH ratio controls sir-tuin protein deacetylase activity, so oxidative metabolism, which drives conversion of NADH to NAD, enhances sirtuin action CR increases mitochondrial biogenesis in liver, fat, and muscle, a response that would raise the NAD/NADH ratio SIRT1 is a key regulator in CR The phys-iological responses caused by CR are the result of a tightly regulated program that connects nutrient availability to the expression of meta-bolic genes A striking feature of CR is the loss of fat stores and reduc-tion in white adipose tissue SIRT1 binding to PPAR, a nuclear

hor-mone receptor that activates transcription of genes involved in adipogenesis and fat storage, represses transcription of these genes, leading to the loss of fat stores Because adipose tissue functions as an endocrine organ, this loss of fat has significant hormonal consequences for energy metabolism In liver, SIRT1 interacts with and deacetylates PGC-1, a transcriptional regulator of glucose production Transcription

of genes encoding the enzymes of gluconeogenesis and repression of genes encoding glycolytic enzymes are increased upon PGC-1 deacety-lation Thus, SIRT1 connects nutrient availability to the regulation of major pathways of energy storage and fuel use

Resveratrol, a phytoalexin, has the same life-extending effects as CR Resveratrol activates both SIRT1 activity and brain AMPK, a key energy sensor The influences of resveratrol on longevity may arise through its effects on caloric homeostasis

PROBLEMS

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1.(Integrates with Chapters 3, 18, and 22.) The conversion of PEP

to pyruvate by pyruvate kinase (glycolysis) and the reverse

reac-tion to form PEP from pyruvate by pyruvate carboxylase and PEP

carboxykinase (gluconeogenesis) represent a so-called substrate

cycle The direction of net conversion is determined by the

rela-tive concentrations of allosteric regulators that exert kinetic

con-trol over pyruvate kinase, pyruvate carboxylase, and PEP

car-boxykinase Recall that the last step in glycolysis is catalyzed by

pyruvate kinase:

PEP ADP 34 pyruvate  ATP The standard free energy change is 31.7 kJ/mol

a Calculate the equilibrium constant for this reaction

b If [ATP] [ADP], by what factor must [pyruvate] exceed [PEP]

for this reaction to proceed in the reverse direction?

The reversal of this reaction in eukaryotic cells is essential to

gluco-neogenesis and proceeds in two steps, each requiring an equivalent

of nucleoside triphosphate energy:

Pyruvate carboxylase

Pyruvate CO2 ATP ⎯⎯→ oxaloacetate  ADP  Pi

PEP carboxykinase

Oxaloacetate GTP ⎯⎯→ PEP  CO2 GDP

Net: Pyruvate ATP  GTP ⎯⎯→ PEP  ADP  GDP  Pi

c The G° for the overall reaction is 0.8 kJ/mol What is the value

of Keq?

d Assuming [ATP] [ADP], [GTP]  [GDP], and Pi 1 mM

when this reaction reaches equilibrium, what is the ratio of

[PEP]/[pyruvate]?

e Are both directions in the substrate cycle likely to be strongly

favored under physiological conditions?

2. (Integrates with Chapter 3.) Assume the following intracellular con-centrations in muscle tissue: ATP 8 mM, ADP  0.9 mM, AMP  0.04 mM, Pi 8 mM What is the energy charge in muscle? What is the

phosphorylation potential?

3. Strenuous muscle exertion (as in the 100-meter dash) rapidly depletes

ATP levels How long will 8 mM ATP last if 1 gram of muscle consumes

300 contains phosphocreatine as a reserve of phosphorylation potential Assuming [phosphocreatine] 40 mM, [creatine]  4 mM, and

G° (phosphocreatine  H2O34 creatine  Pi) 43.3 kJ/mol, how low must [ATP] become before it can be replenished by the reaction: phosphocreatine  ADP 34 ATP  creatine? [Remember,

G° (ATP hydrolysis)  30.5 kJ/mol.]

4. (Integrates with Chapter 20.) The standard reduction potentials for the (NAD/NADH) and (NADP/NADPH) couples are identical, namely, 320 mV Assuming the in vivo concentration ratios NAD/ NADH 20 and NADP/NADPH 0.1, what is G for the

follow-ing reaction?

NADPH NAD34 NADP NADH Assuming standard state conditions for the reaction, ADP  Pi⎯ ATP  H2O, calculate how many ATP equivalents can be formed from ADP  Piby the energy released in this reaction

5. (Integrates with Chapter 3.) Assume the total intracellular pool of adenylates (ATP  ADP  AMP)  8 mM, 90% of which is ATP What are [ADP] and [AMP] if the adenylate kinase reaction is at equilibrium? Suppose [ATP] drops suddenly by 10% What are the concentrations now for ADP and AMP, assuming that the adenylate kinase reaction is at equilibrium? By what factor has the AMP con-centration changed?

6. (Integrates with Chapters 18 and 22.) The reactions catalyzed by PFK and FBPase constitute another substrate cycle PFK is AMP

ac-860 Chapter 27 Metabolic Integration and Organ Specialization

tivated; FBPase is AMP inhibited In muscle, the maximal activity of

PFK (mmol of substrate transformed per minute) is ten times

greater than FBPase activity If the increase in [AMP] described in

problem 5 raised PFK activity from 10% to 90% of its maximal value

but lowered FBPase activity from 90% to 10% of its maximal value,

by what factor is the flux of fructose-6-P through the glycolytic

path-way changed? (Hint: Let PFK maximal activity 10, FBPase

maxi-mal activity 1; calculate the relative activities of the two enzymes

at low [AMP] and at high [AMP]; let J, the flux of F-6-P through the

substrate cycle under any condition, equal the velocity of the PFK

reaction minus the velocity of the FBPase reaction.)

7. (Integrates with Chapters 23 and 24.) Leptin not only induces

syn-thesis of fatty acid oxidation enzymes and uncoupling protein 2 in

adipocytes, but it also causes inhibition of acetyl-CoA carboxylase,

resulting in a decline in fatty acid biosynthesis This effect on

acetyl-CoA carboxylase, as an additional consequence, enhances fatty acid

oxidation Explain how leptin-induced inhibition of acetyl-CoA

car-boxylase might promote fatty acid oxidation

8. (Integrates with Chapters 19 and 20.) Acetate produced in ethanol

metabolism can be transformed into acetyl-CoA by the acetyl

thio-kinase reaction:

Acetate ATP  CoASH ⎯⎯→ acetyl-CoA  AMP  PPi

Acetyl-CoA then can enter the citric acid cycle and undergo

oxida-tion to 2 CO2 How many ATP equivalents can be generated in a

liver cell from the oxidation of one molecule of ethanol to 2 CO2by

this route, assuming oxidative phosphorylation is part of the

process? (Assume all reactions prior to acetyl-CoA entering the

cit-ric acid cycle occur outside the mitochondrion.) Per carbon atom,

which is a better metabolic fuel, ethanol or glucose? That is, how

many ATP equivalents per carbon atom are generated by

combus-tion of glucose versus ethanol to CO2?

9. (Integrates with Chapter 23.) Assuming each NADH is worth 3 ATP,

each FADH2 is worth 2 ATP, and each NADPH is worth 4 ATP:

How many ATP equivalents are produced when one molecule of

palmitoyl-CoA is oxidized to 8 molecules of acetyl-CoA by the fatty

acid -oxidation pathway? How many ATP equivalents are

con-sumed when 8 molecules of acetyl-CoA are transformed into one

molecule of palmitoyl-CoA by the fatty acid biosynthetic pathway?

Can both of these metabolic sequences be metabolically favorable

at the same time if G for ATP synthesis is 50 kJ/mol?

10. (Integrates with Chapters 18–21.) If each NADH is worth 3 ATP,

each FADH2is worth 2 ATP, and each NADPH is worth 4 ATP,

cal-culate the equilibrium constant for cellular respiration, assuming

synthesis of each ATP costs 50 kJ/mol of energy Calculate the

equi-librium constant for CO2fixation under the same conditions,

ex-cept here ATP will be hydrolyzed to ADP  Piwith the release of

50 kJ/mol Comment on whether these reactions are

thermody-namically favorable under such conditions

11. (Integrates with Chapter 22.) In type 2 diabetics, glucose

produc-tion in the liver is not appropriately regulated, so glucose is

over-produced One strategy to treat this disease focuses on the

devel-opment of drugs targeted against regulated steps in glycogenolysis

and gluconeogenesis, the pathways by which liver produces glucose

for release into the blood Which enzymes would you select for as

potential targets for such drugs?

12. As chief scientist for drug development at PhatFarmaceuticals, Inc.,

you want to create a series of new diet drugs You have a grand plan

to design drugs that might limit production of some hormones or

promote the production of others Which hormones are on your

“limit production” list and which are on your “raise levels” list?

13. The existence of leptin was revealed when the ob/ob genetically

obese strain of mice was discovered These mice have a defective

leptin gene Predict the effects of daily leptin injections into ob/ob

mice on food intake, fatty acid oxidation, and body weight Similar clinical trials have been conducted on humans, with limited success Suggest a reason why this therapy might not be a miracle cure for overweight individuals

14.Would it be appropriate to call neuropeptide Y (NPY ) the obesity-promoting hormone? What would be the phenotype of a mouse whose melanocortin-producing neurons failed to produce melano-cortin? What would be the phenotype of a mouse lacking a func-tional MC3R gene? What would be the phenotype of a mouse lack-ing a functional leptin receptor gene?

15.The Human Biochemistry box, The Metabolic Effects of Alcohol Consumption, points out that ethanol is metabolized to acetate in the liver by alcohol dehydrogenase and aldehyde dehydrogenase:

CH3CH2OH NAD34 CH3CHO NADH  H

CH3CHO NAD H2O34 CH3COO NADH  2H

These reactions alter the NAD/NADH ratio in liver cells From your knowledge of glycolysis, gluconeogenesis, and fatty acid oxida-tion, what might be the effect of an altered NAD/NADH ratio on these pathways? What is the basis of this effect?

16.A T172D mutant of the AMPK is locked in a permanently active state Explain

17. a Some scientists support the “malonyl-CoA hypothesis,” which suggests that malonyl-CoA is a key indicator of nutrient avail-ability and the brain uses its abundance to assess whole-body en-ergy homeostasis Others have pointed out that malonyl-CoA is a significant inhibitor of carnitine acyltransferase-1 (see Figure 24.16) Thus, malonyl-CoA may be influencing the levels of an-other metabolite whose concentration is more important as a sig-nal of energy status What metabolite might that be?

b Another test of the malonyl-CoA hypothesis was conducted through the creation of a transgenic strain of mice that lacked functional hypothalamic fatty acid synthase (see Chapter 24) Pre-dict the effect of this genetic modification on cellular malonyl-CoA levels in the hypothalamus, the eating behavior of these transgenic mice, their body fat content, and their physical activity levels Defend your predictions

18. a Leptin was discovered when a congenitally obese strain of mice

(ob/ob mice) was found to lack both copies of a gene encoding a

peptide hormone produced mainly by adipose tissue The pep-tide hormone was named leptin Leptin is an anorexic (appetite-suppressing) agent; its absence leads to obesity Propose an ex-periment to test these ideas

b A second strain of obese mice (db/db mice) produces leptin in abundance but fails to respond to it Assuming the db mutation

leads to loss of function in a protein, what protein is likely to be nonfunctional or absent? How might you test your idea?

Preparing for the MCAT Exam

19.Consult Figure 27.7 and answer the following questions: Which or-gans use both fatty acids and glucose as a fuel in the well-fed state, which rely mostly on glucose, which rely mostly on fatty acids, which one never uses fatty acids, and which one produces lactate

20.Figure 27.3 illustrates the response of R (ATP-regenerating) and U (ATP-utilizing) enzymes to energy charge

a Would hexokinase be an R enzyme or a U enzyme? Would glu-tamine⬊PRPP amidotransferase, the second enzyme in purine biosynthesis, be an R enzyme or a U enzyme?

b If energy charge 0.5: Is the activity of hexokinase high or low? Is ribose-5-P pyrophosphokinase activity high or low?

c If energy charge 0.95: Is the activity of hexokinase high or low?

Is ribose-5-P pyrophosphokinase activity high or low?

FURTHER READING

Systems Analysis of Metabolism

Brand, M D., and Curtis, R K., 2002 Simplifying metabolic complexity

Biochemical Society Transactions 30:25–30.

ATP Coupling and the Thermodynamics of Metabolism

Atkinson, D F., 1977 Cellular Energy Metabolism and Its Regulation New

York: Academic Press

Newsholme, E A., Challiss, R A J., and Crabtree, B., 1984 Substrate

cycles: Their role in improving sensitivity in metabolic control

Trends in Biochemical Sciences 9:277–280.

Newsholme, E A., and Leech, A R., 1983 Biochemistry for the Medical

Sci-ences New York: John Wiley & Sons.

AMP-Activated Protein Kinase

Hardie, D G., 2007 AMP-activated/SNF 1 protein kinases: Conserved

guardians of cellular energy Nature Reviews Cell Molecular Biology

8:774–785

Hardie, D G., Hawley, S A., and Scott, J W., 2007 AMP-activated

pro-tein kinase: Development of the energy sensor concept Journal of

Physiology 574:7–15.

McGee, S L., and Hargreaves, M., 2008 AMPK and transcriptional

reg-ulation Frontiers in Bioscience 13:3022–3033.

Metabolic Relationships Between Organ Systems

Harris, R., and Crabb, D W., 1997 Metabolic interrelationships In

Text-book of Biochemistry with Clinical Correlations, 4th ed., Devlin, T M., ed.

New York: Wiley-Liss

Sugden, M C., Holness, M J., and Palmer, T N., 1989 Fuel selection

and carbon flux during the starved-to-fed transition Biochemical

Journal 263:313–323.

Creatine as a Nutritional Supplement

Ekblom, B., 1999 Effects of creatine supplementation on performance

American Journal of Sports Medicine 24:S-38.

Kreider, R., 1998 Creatine supplementation: Analysis of ergogenic

value, medical safety, and concerns Journal of Exercise Physiology 1,

an international onnline journal available at http://www.css.edu/

users/tboone2/asep/jan3.htm

Fat-Free Mice

Gavrilova, O., et al., 2000 Surgical implantation of adipose tissue

re-verses diabetes in lipoatrophic mice Journal of Clinical Investigation

105:271–278

Moitra, J., et al., 1998 Life without white fat: A transgenic mouse Genes

and Development 12:3168–3181.

Leptin and Hormonal Regulation of Eating Behavior

Barinaga, M., 1995 “Obese” protein slims mice Science 269:475–476,

and references therein

Buettner, C., 2007 Does FASing out new fat in the hypothalamus make

you slim? Cell Metabolism 6:249–251.

Clement, K., et al., 1998 A mutation in the human leptin receptor gene

causes obesity and pituitary dysfunction Nature 392:398–401.

Coll, A P., Farooqi, S., and O’Rahilly, S., 2007 The hormonal control of

food intake Cell 129:251–262.

Saper, C B., Chou, T C., and Elmquist, J K., 2002 The need to feed:

Homeostatic and hedonic control of eating Neuron 36:199–211.

Schwartz, M W., and Morton, G J., 2002 Obesity: Keeping hunger at

bay Nature 418:595–597.

Vaisse, C., et al., 2000 Melanocortin-4 receptor mutations are a

fre-quent and heterogeneous cause of morbid obesity Journal of

Clini-cal Investigation 106:253–262.

Zhou, Y-T., et al., 1997 Induction by leptin of uncoupling protein-2 and

enzymes of fatty acid oxidation Proceedings of the National Academy of

Sciences U.S.A 94:6386–6390.

Caloric Restriction and Longevity

Dasgupta, B., and Milbrandt, J., 2007 Resveratrol stimulates AMP kinase

activity in neurons Proceedings of the National Academy of Science U.S.A.

104:7217–7222

Denu, J M., 2005 The Sir2 family of protein deacetylases Current

Opin-ion in Chemical Biology 9:431–440.

Guarente, L., 2005 Caloric restriction and SIR2 genes—Towards a

mechanism Mechanisms of Aging and Development 126:923–928.

Guarente, L., and Picard, F., 2005 Caloric restriction—The SIR2

con-nection Cell 120:473–482.

Michan, S., and Sinclair, D., 2007 Sirtuins in mammals: Insights into

their biological function Biochemical Journal 404:1–13.

Milne, J C., Lambert, P D., Schenk, S., Carney, D P., et al., 2007 Small molecule activators of SIRT1 as therapeutics for the treatment of

type 2 diabetes Nature 450:712–716.

Moynihan, K A., and Imai, S-I., 2006 Sirt1 as a key regulator

orches-trating the response to caloric restriction Drug Discovery Today:

Dis-ease Mechanisms 3:11–17.

“Dawn of the Double Helix,” by Julie Newdoll

28 DNA Metabolism: Replication,

Recombination, and Repair

Heredity, which we can define generally as the tendency of an organism to pos-sess the characteristics of its parent(s), is clearly evident throughout nature and since the dawn of history has served to justify the classification of organisms ac-cording to shared similarities The basis of heredity, however, was a mystery Early

in the 20th century, geneticists demonstrated that genes, the elements or units

carrying and transferring inherited characteristics from parent to offspring, are contained within the nuclei of cells in association with the chromosomes Yet the chemical identity of genes remained unknown, and genetics was an abstract sci-ence Even the realization that chromosomes are composed of proteins and nu-cleic acids did little to define the molecular nature of the gene because, at the time, no one understood either of these substances.

The material of heredity must have certain properties It must be very stable so that genetic information can be stored in it and transmitted countless times to sub-sequent generations It must be capable of precise copying or replication so that its information is not lost or altered And, although stable, it must also be subject to change in order to account, in the short term, for the appearance of mutant forms and, in the long term, for evolution DNA is the material of heredity.

28.1 How Is DNA Replicated?

Transfer of genetic information from generation to generation requires the faithful reproduction of the parental DNA DNA reproduction produces two identical

copies of the original DNA in a process termed DNA replication The mechanism

for DNA replication is strand separation followed by the copying of each strand In the

process, each separated strand acts as a template for the synthesis of a new

com-plementary strand whose nucleotide sequence is fixed by the base-pairing rules Watson and Crick proposed (see Chapter 10) Strand separation is achieved by untwisting the double helix (Figure 28.1) Base pairing then dictates the proper sequence of nucleotide addition to achieve an accurate replication of each orig-inal strand Thus, each origorig-inal strand ends up paired with a new complementary partner, and two identical double-stranded DNA molecules are formed from one.

This mode of DNA replication is referred to as semiconservative because one of

the two original strands is conserved in each of the two progeny molecules.

DNA Replication Is Bidirectional

Replication of DNA molecules begins at one or more specific regions called the

origin(s) of replication and, excepting certain bacteriophage chromosomes and plas-mids, proceeds in both directions from this origin (Figure 28.2) For example,

repli-cation of E coli DNA begins at oriC, a unique 245-bp chromosomal site that contains

11 GATC tetranucleotide sequences along its length From oriC, replication

ad-vances in both directions around the circular chromosome That is, bidirectional

Julie Newdoll’s painting “Dawn of the Double Helix”

composes the DNA duplex as human figures Her

theme in this painting is “Life Forms: The basic

struc-tures that make our existence possible.”

Heredity

I am the family face;

Flesh perishes, I live on,

Projecting trait and trace

Through time to times anon,

And leaping from place to place

Over oblivion.

The years-heired feature that can

In curve and voice and eye

Despise the human span

Of durance—that is I;

The eternal thing in man,

That heeds no call to die.

Thomas Hardy

(in Moments of Vision and Miscellaneous Verses, 1917)

KEY QUESTIONS

28.1 How Is DNA Replicated?

28.2 What Are the Properties of DNA

Polymerases?

28.3 Why Are There So Many DNA Polymerases?

28.4 How Is DNA Replicated in Eukaryotic Cells?

28.5 How Are the Ends of Chromosomes

Replicated?

28.6 How Are RNA Genomes Replicated?

28.7 How Is the Genetic Information Shuffled

by Genetic Recombination?

28.8 Can DNA Be Repaired?

28.9 What Is the Molecular Basis of Mutation?

28.10 Do Proteins Ever Behave as Genetic Agents?

Special Focus: Gene Rearrangements

and Immunology—Is It Possible to

Generate Protein Diversity Using Genetic

Recombination?

ESSENTIAL QUESTIONS

DNA is the physical repository of genetic information in the cell and the material of heredity that is passed on to progeny.

How is this genetic information in the form of DNA replicated, how is the infor-mation rearranged, and how is its integrity maintained in the face of damage?

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