The second regulatory site, the substrate specificity site, can bind either ATP, dTTP, dGTP, or dATP, and the substrate specificity of the enzyme is determined by which of these nucleoti
Trang 1enzyme must be turned on and off in response to the need for dNTPs Second,
the relative amounts of each NDP substrate transformed into dNDP must be
con-trolled so that the right balance of dATP⬊dGTP⬊dCTP⬊dTTP is produced The
two different effector-binding sites on ribonucleotide reductase, discrete from the
substrate-binding catalytic site, are designed to serve these purposes As noted
previ-ously, these two regulatory sites are designated the overall activity site and the
sub-strate specificity site Only ATP and dATP are able to bind at the overall activity site.
ATP is an allosteric activator and dATP is an allosteric inhibitor, and they compete
for the same site If ATP is bound, the enzyme is active, whereas if its deoxy
coun-terpart, dATP, occupies this site, the enzyme is inactive That is, ATP is a positive
effector and dATP is a negative effector with respect to enzyme activity, and they
compete for the same site.
The second regulatory site, the substrate specificity site, can bind either ATP,
dTTP, dGTP, or dATP, and the substrate specificity of the enzyme is determined
by which of these nucleotides occupies this site If ATP is in the substrate specificity
site, ribonucleotide reductase preferentially binds pyrimidine nucleotides (UDP
or CDP) at its active site and reduces them to dUDP and dCDP With dTTP in the
specificity-determining site, GDP is the preferred substrate When dGTP binds to
the specificity site, ADP becomes the favored substrate for reduction The
ratio-nale for these varying affinities is as follows (Figure 26.23): High [ATP] is
consis-tent with cell growth and division and, consequently, the need for DNA synthesis.
Thus, ATP binds in the overall activity site of ribonucleotide reductase, turning it
on and promoting production of dNTPs for DNA synthesis Under these
condi-tions, ATP is also likely to occupy the substrate specificity site, so UDP and CDP are
bound at the catalytic site and reduced to dUDP and dCDP Both of these
pyrimi-dine deoxynucleoside diphosphates are precursors to dTTP Thus, elevation of
dUDP and dCDP levels leads to an increase in [dTTP] High dTTP levels increase
the likelihood that it will occupy the substrate specificity site, in which case GDP
be-comes the preferred substrate and dGTP levels rise Upon dGTP association with
the substrate specificity site, ADP is the favored substrate, leading to ADP reduction
and the eventual accumulation of dATP Binding of dATP to the overall activity site
then shuts the enzyme down In summary, the relative affinities of the three
classes of nucleotide binding sites in ribonucleotide reductase for the various
sub-strates, activators, and inhibitors are such that the formation of dNDPs proceeds
in an orderly and balanced fashion As these dNDPs are formed in amounts
con-sistent with cellular needs, their phosphorylation by nucleoside diphosphate
ki-nases produces dNTPs, the actual substrates of DNA synthesis.
26.8 How Are Thymine Nucleotides Synthesized?
The synthesis of thymine nucleotides proceeds from other pyrimidine
deoxyri-bonucleotides Cells have no requirement for free thymine ribonucleotides and
do not synthesize them Small amounts of thymine ribonucleotides do occur in
tRNA (tRNA is notable for having unusual nucleotides), but these Ts arise via
methylation of U residues already incorporated into the tRNA Both dUDP and
1
2
3
4
5
6
Energy status of cell is robust; [ATP] is high Make DNA:
ATP occupies activity site A: ribonucleotide reductase ON
ATP in specificity site S favors CDP or UDP in catalytic site C [dCDP], [dUDP]
dCDP
dTTP occupies specificity site S, favoring GDP or ADP in catalytic site C
dGTP occupies specificity site S, favoring ADP in catalytic site C [dADP]
dATP replaces ATP in activity site A: ribonucleotide reductase OFF
GDP dGDP dGTP
FIGURE 26.23 Regulation of deoxynucleotide biosyn-thesis: the rationale for the various affinities displayed
by the two nucleotide-binding regulatory sites on ribo-nucleotide reductase
Trang 2834 Chapter 26 Synthesis and Degradation of Nucleotides
dCDP can lead to formation of dUMP, the immediate precursor for dTMP syn-thesis (Figure 26.24) Interestingly, formation of dUMP from dUDP passes
through dUTP, which is then cleaved by dUTPase, a pyrophosphatase that
re-moves PPifrom dUTP The action of dUTPase prevents dUTP from serving as a substrate in DNA synthesis An alternative route to dUMP formation starts with
From dUDP: dUDP From dCDP: dCDP
dUTP dCMP
dUMP dUMP
dTMP dTMP
FIGURE 26.24 Pathways of dTMP synthesis dTMP
pro-duction is dependent on dUMP formation from dCDP
and dUDP
A DEEPER LOOK
Fluoro-Substituted Analogs as Therapeutic Agents
Carbon–fluorine bonds are exceedingly rare in nature, and
fluo-rine is an uncommon constituent of biological molecules F has
three properties attractive to drug designers: (1) It is the smallest
replacement for an H atom in organic synthesis, (2) fluorine is
the most electronegative element, and (3) the FOC bond is
rela-tively unreactive This steric compactness and potential for strong
inductive effects through its electronegativity renders F a useful
substituent in the construction of inhibitory analogs of enzyme
substrates One interesting strategy is to devise fluorinated
pre-cursors that are taken up and processed by normal metabolic
pathways to generate a potent antimetabolite A classic example is
fluoroacetate FCH2COOis exceptionally toxic because it is
read-ily converted to fluorocitrate by citrate synthase of the citric acid
cycle (see Chapter 19) In turn, fluorocitrate is a powerful
in-hibitor of aconitase The metabolic transformation of an
other-wise innocuous compound into a poisonous derivative is termed
lethal synthesis 5-Fluorouracil and 5-fluorocytosine are also
exam-ples of this strategy (see Human Biochemistry on page 835)
Unlike hydrogen, which is often abstracted from substrates as
H, electronegative fluorine cannot be readily eliminated as the
corresponding F Thus, enzyme inhibitors can be fashioned in
which F replaces H at positions where catalysis involves H removal
as H Thymidylate synthase catalyzes removal of H from dUMP
as Hthrough a covalent catalysis mechanism A thiol group on this
enzyme normally attacks the 6-position of the uracil moiety
of 2-deoxyuridylic acid so that C-5 can act as a carbanion in attack
on the methylene carbon of N5,N10-methylene-THF (see
accompa-nying figure) Regeneration of free enzyme then occurs through
loss of the C-5 H atom as Hand dissociation of product dTMP If
F replaces H at C-5 as in 2-deoxy-5-fluorouridylate (FdUMP), the
enzyme is immobilized in a very stable ternary [enzyme⬊FdUMP⬊
methylene-THF] complex and effectively inactivated Enzyme
in-hibitors like FdUMP whose adverse properties are elicited only
through direct participation in the catalytic cycle are variously
called mechanism-based inhibitors, suicide substrates, or Trojan
horse substrates.
+
dR
dR
dR
H
H2N N
O
N
N
N H H
CH2 N
H2C
R
H
2 3 4 1
8 7
6 9 10 5
N
H
2 3 4
1 6 5
O H
O
Cys SH
N
H
2 3 4
1 6 5
O H
O
–
S Cys
N
N
H
O H
Cys
CH2
F N
H2N N
O
N
N
H
H
CH2 H
2 3 4
6 9
10 5
E
E
E
R H
H
H
N5,N10 -methylene- THF
Ternary complex
䊳 The effect of the 5-fluoro substitution on the mechanism of action
of thymidylate synthase An enzyme thiol group (from a Cys side chain)
ordinarily attacks the 6-position of dUMP so that C-5 can react as a
carbanion with N5,N10-methylene-THF Normally, free enzyme is
regenerated following release of the hydrogen at C-5 as a proton
Because release of fluorine as Fcannot occur, the ternary (three-part)
complex of [enzyme⬊fluorouridylate⬊methylene-THF] is stable and
per-sists, preventing enzyme turnover (The N5,N10-methylene-THF structure
is given in abbreviated form.)
Trang 3dCDP, which is dephosphorylated to dCMP and then deaminated by dCMP
deaminase (Figure 26.25), leaving dUMP dCMP deaminase provides a second
point for allosteric regulation of dNTP synthesis; it is allosterically activated by
dCTP and feedback-inhibited by dTTP Of the four dNTPs, only dCTP does not
interact with either of the regulatory sites on ribonucleotide reductase (see
Fig-ure 26.20) Instead, it acts upon dCMP deaminase.
Synthesis of dTMP from dUMP is catalyzed by thymidylate synthase (Figure
26.26) This enzyme methylates dUMP at the 5-position to create dTMP; the
methyl donor is the one-carbon folic acid derivative N5,N10-methylene-THF The
reaction is actually a reductive methylation in which the one-carbon unit is
trans-ferred at the methylene level of reduction and then reduced to the methyl level.
The THF cofactor is oxidized at the expense of methylene reduction to yield
DHF DHFR then reduces DHF back to THF for service again as a one-carbon
vehicle (see panel a of the figure in A Deeper Look on page 816) Thymidylate
synthase sits at a junction connecting dNTP synthesis with folate metabolism
dCMP deaminase
NH2
O
O
N
N
H
OH
dCMP
NH4
O O
N N
H OH
dUMP
O H
+
H++ H2O
FIGURE 26.25 (a) The dCMP deaminase reaction (b) Trimeric dCMP deaminase Each chain has a bound dCTP
molecule (purple) and a Mg2ion (orange) (pdb id 1XS4)
HUMAN BIOCHEMISTRY
Fluoro-Substituted Pyrimidines in Cancer Chemotherapy, Fungal Infections,
and Malaria
5-Fluorouracil (5-FU; see part a of the figure) is a thymine analog It
is converted in vivo to 5 -fluorouridylate by a PRPP-dependent
phos-phoribosyltransferase and passes through the reactions of dNTP
synthesis, culminating ultimately as 2 -deoxy-5-fluorouridylic acid, a
potent inhibitor of dTMP synthase (see A Deeper Look on page
834) 5-FU is used as a chemotherapeutic agent in the treatment of
human cancers Similarly, 5-fluorocytosine (see part b) is used as an
antifungal drug because fungi, unlike mammals, can convert it to
2-deoxy-5-fluorouridylate Furthermore, malarial parasites can use
exogenous orotate to make pyrimidines for nucleic acid synthesis,
whereas mammals cannot Thus, 5-fluoroorotate (see part c) is an
effective antimalarial drug because it is selectively toxic to these
parasites
NH2
O–
O
H N N
O F
H
O
N N F
H
O
H N N F
H
O
5-Fluorouracil 5-Fluorocytosine
5-Fluoroorotate
(a)
(c)
(b)
Trang 426.1 Can Cells Synthesize Nucleotides? Nucleotides are ubiquitous
constituents of life and nearly all cells are capable of synthesizing them
“from scratch” via de novo pathways Rapidly proliferating cells must
make lots of purine and pyrimidine nucleotides to satisfy demands for
DNA and RNA synthesis Nucleotide biosynthetic pathways are attractive
targets for the clinical control of rapidly dividing cells such as cancers or
infectious bacteria Many antibiotics and anticancer drugs are inhibitors
of purine or pyrimidine nucleotide biosynthesis
26.2 How Do Cells Synthesize Purines? The nine atoms of the purine
ring system are derived from aspartate (N-1), glutamine (N-3 and N-9),
glycine (C-4, C-5, and N-7), CO2(C-6), and THF one-carbon derivatives
(C-2 and C-8) The atoms of the purine ring are successively added to
ribose-5-phosphate, so purines begin as nucleotide derivatives through
as-sembly of the purine ring system directly on the ribose Because purine
biosynthesis depends on folic acid derivatives, it is sensitive to inhibition
by folate analogs Distinct, two-step metabolic pathways diverge from IMP,
one leading to AMP and the other to GMP Purine biosynthesis is regu-lated at several stages: Reaction 1 (ribose-5-phosphate pyrophosphoki-nase) is feedback-inhibited by ADP and GDP; the enzyme catalyzing re-action 2 (glutamine phosphoribosyl pyrophosphate amidotransferase) has two inhibitory allosteric sites, one where adenine nucleotides bind and another where guanine nucleotides bind PRPP is a “feed-forward” activator of this enzyme The first reaction in the conversion of IMP to AMP involves adenylosuccinate synthetase, which is inhibited by AMP; the first step in the conversion of IMP to GMP is catalyzed by IMP dehydro-genase and is inhibited by GMP ATP-dependent kinases form nucleoside diphosphates and triphosphates from AMP and GMP
26.3 Can Cells Salvage Purines? Purine ring systems represent a meta-bolic investment by cells, and salvage pathways exist to recover them when degradation of nucleic acids releases free purines in the form of adenine, guanine, and hypoxanthine (the base in IMP) Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) acts on either
hypoxan-836 Chapter 26 Synthesis and Degradation of Nucleotides
It has become a preferred target for inhibitors designed to disrupt DNA synthe-sis An indirect approach is to employ folic acid precursors or analogs as anti-metabolites of dTMP synthesis (see panel b of the figure in A Deeper Look on page 818) Purine synthesis is affected as well because it is also dependent on THF (see Figure 26.3).
dR
O
O–
H N N O
H
H2N N
O
N
N
N H H
CH2 N
H2C
R
H H
2
7
6
9 10 5
H
H2N N
O
N
N
N
H H
CH2 N H
R
H H
H
H
R O
H N N
O
CH3
H
H2N N
O
N
N
H
CH2 N R H O
C
CH2
+H3N
O C CH +H3N
CH2 OH
N5,N10 -methylene-THF
Thymidylate synthase
Dihydrofolic acid (DHF) Serine
hydroxymethyl-transferase
Dihydrofolate reductase
Tetrahydrofolic acid (THF)
(a)
NADP+
Glycine
NADPH + H+
(b)
FIGURE 26.26 (a) The thymidylate synthase reaction The 5-CH3group is ultimately derived from the -carbon
of serine (b) Thymidylate synthase dimer Each monomer has a bound folate analog (green) and dUMP (light
blue) (pdb id 1JUJ)
Trang 5Preparing for an exam? Create your own study path for this
chapter at www.cengage.com/login
1.Draw the purine and pyrimidine ring structures, indicating the
metabolic source of each atom in the rings
2.Starting from glutamine, aspartate, glycine, CO2 and N10
-formyl-THF, how many ATP equivalents are expended in the synthesis of
(a) ATP, (b) GTP, (c) UTP, and (d) CTP?
3.Illustrate the key points of regulation in (a) the biosynthesis of IMP,
AMP, and GMP; (b) E coli pyrimidine biosynthesis; and (c)
mam-malian pyrimidine biosynthesis
4.Indicate which reactions of purine or pyrimidine metabolism are
af-fected by the inhibitors (a) azaserine, (b) methotrexate, (c)
sulfon-amides, (d) allopurinol, and (e) 5-fluorouracil
5.Since dUTP is not a normal component of DNA, why do you
sup-pose ribonucleotide reductase has the capacity to convert UDP to
dUDP?
6.Describe the underlying rationale for the regulatory effects exerted
on ribonucleotide reductase by ATP, dATP, dTTP, and dGTP
7.(Integrates with Chapters 18–20 and 22.) By what pathway(s) does
the ribose released upon nucleotide degradation enter intermediary
metabolism and become converted to cellular energy? How many
ATP equivalents can be recovered from one equivalent of ribose?
8.(Integrates with Chapter 25.) At which steps does the purine
biosyn-thetic pathway resemble the pathway for biosynthesis of the amino
acid histidine?
9.Write reasonable chemical mechanisms for steps 6, 8, and 9 in
purine biosynthesis (see Figure 26.3)
10.Write a balanced equation for the conversion of aspartate to
fu-marate by the purine nucleoside cycle in skeletal muscle
11.Write a balanced equation for the oxidation of uric acid to glyoxylic
acid, CO2, and NH3, showing each step in the process and naming
all of the enzymes involved
12. (Integrates with Chapter 15.) E coli aspartate transcarbamoylase
(ATCase) displays classic allosteric behavior This 66 enzyme is activated by ATP and feedback-inhibited by CTP In analogy with the behavior of glycogen phosphorylase shown in Figure 15.14,
il-lustrate the allosteric v versus [aspartate] curves for ATCase (a) in
the absence of effectors, (b) in the presence of CTP, and (c) in the presence of ATP
*13. (Integrates with Chapter 15.) Unlike its allosteric counterpart glyco-gen phosphorylase (an 2enzyme), E coli ATCase has a heteromeric
(66) organization The -subunits bind aspartate and are
consid-ered catalytic subunits, whereas the -subunits bind CTP or ATP and
are considered regulatory subunits How would you describe the sub-unit organization of ATCase from a functional point of view?
14. (Integrates with Chapter 20.) Starting from HCO3 , glutamine, as-partate, and ribose-5-P, how many ATP equivalents are consumed in the synthesis of dTTP in a eukaryotic cell, assuming dihydroorotate oxidation is coupled to oxidative phosphorylation? How does this result compare with the ATP costs of purine nucleotide biosynthe-sis calculated in problem 2?
15. Write a balanced equation for the synthesis of dTMP from UMP and
N5,N10-methylene-THF Thymidylate synthase has four active-site arginine residues (Arg23, Arg178, Arg179, and Arg218) involved in substrate binding Postulate a role for the side chains of these Arg residues
16. Enzymes that bind phosphoribosyl-5-phosphate (PRPP) have a com-mon structural fold, the PRT fold, which unites them as a structural family PRT here refers to the phosphoribosyl transferase activity dis-played by some family members Typically, in such reactions, PPiis displaced from PRPP by a nitrogen-containing nucleophile Several such reactions occur in purine metabolism Identify two such reac-tions where the enzyme involved is likely to be a PRT family member
17. The crystal structure of E coli dihydrofolate reductase (DFR) with
NADPand folate bound can be found in the Protein Data Bank
thine to form IMP or guanine to form GMP; an absence of HGPRT is the
basis of Lesch-Nyhan syndrome
26.4 How Are Purines Degraded? Dietary nucleic acids are digested
to nucleotides by various nucleases and phosphodiesterases, the
nu-cleotides are converted to nucleosides by base-specific nucleotidases
and nonspecific phosphatases, and then nucleosides are hydrolyzed to
release the purine base Only the pentoses of nucleotides serve as
sources of metabolic energy In humans, the purine ring is oxidized to
uric acid by xanthine oxidase and excreted Gout occurs when bodily
fluids accumulate an excess of uric acid Skeletal muscle operates a
purine nucleoside cycle as an anaplerotic pathway
26.5 How Do Cells Synthesize Pyrimidines? In contrast to formation of
the purine ring system, the pyrimidine ring system is completed before a
ribose-5-P moiety is attached Only two precursors, carbamoyl-P and
as-partate, contribute atoms to the six-membered pyrimidine ring The first
step in humans is catalyzed by CPS-II ATCase then links carbamoyl-P with
aspartate Subsequent reactions close the ring and oxidize it before
adding ribose-5-P, using -PRPP as donor Decarboxylation gives UMP In
mammals, the six enzymatic activities of pyrimidine biosynthesis are
dis-tributed among only three proteins, two of which are multifunctional
polypeptides Purine and pyrimidine synthesis in mammals are two
prominent examples of metabolic channeling UMP leads to UTP, the
substrate for formation of CTP via CTP synthetase Regulation of
pyrimi-dine synthesis in animals occurs at CPS-II UDP and UTP are feedback
inhibitors, whereas PRPP and ATP are allosteric activators In bacteria,
regulation acts at ATCase through feedback inhibition by CTP (or UTP)
and activation by ATP
26.6 How Are Pyrimidines Degraded? Degradation of the pyrimi-dine ring generates -alanine, CO2, and ammonia In humans, pyrim-idines are recycled from nucleosides, but free pyrimidine bases are not salvaged
26.7 How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis? 2-Deoxyribonucleotides are formed from ribonu-cleotides through reduction at the 2-position of the ribose ring in NDPs The reaction, catalyzed by ribonucleotide reductase, involves a free radi-cal mechanism that replaces the 2-OH by a hydride ion (H⬊) Thiore-doxin provides the reducing power for ribonucleotide reduction Class Ia ribonucleotide reductases have three different nucleotide-binding sites: the catalytic site (or active site), which binds substrates (ADP, CDP, GDP, and UDP); the substrate specificity site, which can bind ATP, dATP, dGTP,
or dTTP; and the overall activity site, which binds either the activator ATP
or the negative effector dATP The relative affinities of the three classes of nucleotide binding sites in ribonucleotide reductase for the various sub-strates, activators, and inhibitors are such that the various dNDPs are formed in amounts consistent with cellular needs
26.8 How Are Thymine Nucleotides Synthesized? Both dUDP and dCDP can lead to formation of dUMP, the immediate precursor for dTMP synthesis Formation of dTMP from dUMP is catalyzed by thymidylate syn-thase through reductive methylation of dUMP at the 5-position The
methyl donor is the one-carbon folic acid derivative N5,N10 -methylene-THF Fluoro-substituted pyrimidine analogs such as 5-fluorouracil (5-FU), 5-fluorocytosine, and 5-fluoroorotate can be converted to FdUMP, which inhibits thymidylate synthase These fluoro compounds have found a range of therapeutic uses in treating diseases from cancer to malaria
Trang 6838 Chapter 26 Synthesis and Degradation of Nucleotides
(www.rcsb.org/pdb) as file 7DFR Go to this website, enter “7DFR”
in the search line, and click on “KiNG” under “Display options”
when the 7DFR page comes up Explore the KiNG graphic of the
DFR structure to visualize how its substrates are bound (If you hold
down the left button on your mouse and move the cursor over the
image, you can rotate the structure to view it from different
per-spectives.) Note in particular the spatial relationship between the
nicotinamide ring of NADPand the pterin ring of folate Do you
now have a better appreciation for how this enzyme works? Note
also the location of polar groups on the two substrates in relation to
the DFR structure
18.E coli aspartate transcarbamoylase is an allosteric enzyme (see
problem 12) composed of six catalytic (C) subunits and six
regula-tory (R) subunits Protein Data Bank file 1RAA shows one-third of
the ATCase holoenzyme (two C subunits and two R subunits; CTP
molecules are bound to the R subunits) Explore this structure
us-ing the KiNG display option What secondary structural motif
dom-inates the R subunit structure? Protein Data Bank file 2IPO also
shows one-third of the ATCase holoenzyme (two C subunits and two R subunits), but in this structure molecules of the substrate
analog N-(2-phosphonoacetyl)-L-asparagine are bound to the C
subunits Explore this structure using the KiNG display option Note the distance separating the ATCase active site from its al-losteric site Interpret what you see in terms of the Monod– Wyman–Changeux model for allosteric regulation (see Chapter 15) Which of these structures corresponds to the MWC R-state, and which corresponds to the T-state?
Preparing for the MCAT Exam
19.Examine Figure 26.6 and predict the relative rates of the regulated reactions in the purine biosynthetic pathway from ribose-5-P to GMP and AMP under conditions in which GMP levels are very high
20.Decide from Figures 18.1, 25.31, 26.26, and the Deeper Look box
on page 817 which carbon atom(s) in glucose would be most likely
to end up as the 5-CH3carbon in dTMP
FURTHER READING
Purine Metabolism
Kisker, C., Schindelin, H., and Rees, D C., 1997 Molybdenum-containing
enzymes: Structure and mechanism Annual Review of Biochemistry
66:233–267
Mueller, E J., et al., 1994 N5-carboxyaminoimidazole ribonucleotide:
Evidence for a new intermediate and two new enzymatic activities in
the de novo purine biosynthetic pathway of Escherichia coli
Biochem-istry 33:2269–2278.
Watts, R W E., 1983 Some regulatory and integrative aspects of purine
nucleotide synthesis and its control: An overview Advances in Enzyme
Regulation 21:33–51.
Wilson, D K., Rudolph, F B., and Quiocho, F A., 1991 Atomic
struc-ture of adenosine deaminase complexed with a transition-state
analog: Understanding catalysis and immunodeficient mutations
Science 252:1279–1284.
Pyrimidine Metabolism
Connolly, G P., and Duley, J A., 1999 Uridine and its nucleotides:
Bio-logical actions, therapeutical potentials Trends in PhrmacoBio-logical
Sci-ences 20:218–225.
Graves, L M., et al., 2000 Regulation of carbamoyl phosphate
synthe-tase by MAP kinase Nature 403:328–331.
Jones, M E., 1980 Pyrimidine nucleotide biosynthesis in animals: Genes,
enzymes and regulation of UMP biosynthesis Annual Review of
Bio-chemistry 49:253–279.
Metabolic Disorders of Purine and Pyrimidine Metabolism
Löffler, M., et al., 2005 Pyrimidine pathways in health and disease
Trends in Molecular Medicine 11:430–437.
Nyhan, W L., 2005 Disorders of purine and pyrimidine metabolism
Molecular Genetics and Metabolism 86:25–33.
Scriver, C R., et al., 1995 The Metabolic Bases of Inherited Disease, 7th ed.
New York: McGraw-Hill
Metabolic Channeling
Benkovic, S J., 1984 The transformylase enzymes in de novo purine
biosynthesis Trends in Biochemical Sciences 9:320–322.
Henikoff, S., 1987 Multifunctional polypeptides for purine de novo
syn-thesis BioEssays 6:8–13.
Huang, X., Holden, H M., and Raushel, F M., 2001 Channeling of
sub-strates and intermediates in enzyme-catalyzed reactions Annual
Re-view of Biochemistry 70:149–180.
Srere, P A., 1987 complexes of sequential metabolic enzymes Annual
Review of Biochemistry 56:89–124.
Deoxyribonucleotide Biosynthesis
Carreras, C W., and Santi, D V., 1995 The catalytic mechanism and
struc-ture of thymidylate synthase Annual Review of Biochemistry 64:721–762.
Frey, P A., 2001 Radical mechanisms of enzymatic catalysis Annual
Re-view of Biochemistry 70:121–148.
Herrick, J., and Sciavi, B., 2007 Ribonucleotide reductase and the reg-ulation of DNA replication: An old story and an ancient heritage
Molecular Microbiology 63:22–34.
Jordan, A., and Reichard, P., 1998 Ribonucleotide reductases Annual
Review of Biochemistry 67:71–98.
Licht, S., Gerfen, G J., and Stubbe, J., 1996 Thiyl radicals in
ribonu-cleotide reductases Science 271:477–481.
Marsh, E N G., 1995 A radical approach to enzyme catalysis BioEssays
17:431–441
Reichard, P., 1988 Interactions between deoxyribonucleotide and DNA
synthesis Annual Review of Biochemistry 57:349–374.
Stubbe, J., Ge, J., and Yee, C S., 2001 The evolution of ribonucleotide
reduction revisited Trends in Biochemical Sciences 26:93–99.
Inhibitors of Purine, Pyrimidine, and Deoxyribonucleotide Biosynthesis as Therapeutic Agents
Abeles, R H., and Alston, T A., 1990 Enzyme inhibition by fluoro
com-pounds Journal of Biological Chemistry 265:16705–16708.
Galmarini, C M., Mackey, J R., and Dumontet, C., 2002 Nucleoside
analogues and nucleobases in cancer treatment Lancet Oncology
3:415–424
Hitchings, G H., 1992 Antagonists of nucleic acid derivatives as
medic-inal agents Annual Review of Pharmacology and Toxicology 32:1–9.
Park, B K., Kitteringham, N R., and O’Neill, P M., 2001 Metabolism of
fluorine-containing drugs Annual Review of Pharmacology and
Toxi-cology 41:443–470.
Zrenner, R., et al., 2006 Pyrimidine and purine biosynthesis and
degra-dation in plants Annual Review of Plant Biology 57:805–836.
Trang 7Copyright © 2008 W
and Organ Specialization
In the preceding chapters, we have explored the major metabolic pathways—
glycolysis, the citric acid cycle, electron transport and oxidative phosphorylation,
photosynthesis, gluconeogenesis, fatty acid oxidation, lipid biosynthesis, amino
acid metabolism, and nucleotide metabolism Several of these pathways are
cata-bolic and serve to generate chemical energy useful to the cell; others are anacata-bolic
and use this energy to drive the synthesis of essential biomolecules Despite their
opposing purposes, these reactions typically occur at the same time as nutrient
molecules are broken down to provide the building blocks and energy for ongoing
biosynthesis Cells maintain a dynamic steady state through processes that involve
considerable metabolic flux The metabolism that takes place in just a single cell is
so complex that it defies detailed quantitative description However, an
apprecia-tion of overall relaapprecia-tionships can be achieved by stepping back and considering
in-termediary metabolism at a systems level of organization.
27.1 Can Systems Analysis Simplify the Complexity
of Metabolism?
The metabolism of a typical heterotrophic cell can be portrayed by a schematic
diagram consisting of just three interconnected functional blocks: (1) catabolism,
(2) anabolism, and (3) macromolecular synthesis and growth (Figure 27.1).
1 Catabolism Energy-yielding nutrients are oxidized to CO2and H2O in
catabo-lism, and most of the electrons liberated are passed to oxygen via an
electron-transport pathway coupled to oxidative phosphorylation, resulting in the
forma-tion of ATP Some electrons go to reduce NADP to NADPH, the source of
reducing power for anabolism Glycolysis, the citric acid cycle, electron transport
and oxidative phosphorylation, and the pentose phosphate pathway are the
prin-cipal pathways within this block The metabolic intermediates in these pathways
also serve as substrates for processes within the anabolic block.
2 Anabolism The biosynthetic reactions that form the many cellular molecules
collectively comprise anabolism For thermodynamic reasons, the chemistry of
an-abolism is more complex than that of catan-abolism (that is, it takes more energy [and
often more steps] to synthesize a molecule than can be produced from its
degrada-tion) Metabolic intermediates derived from glycolysis and the citric acid cycle are
the precursors for this synthesis, with NADPH supplying the reducing power and
ATP the coupling energy.
3 Macromolecular Synthesis and Growth The organic molecules produced in
anabolism are the building blocks for creation of macromolecules Like anabolism,
The Washington, D.C., Metro map The coordinated flow of passengers along different transit lines is an apt metaphor for metabolic regulation
Study of an enzyme, a reaction, or a sequence can
be biologically relevant only if its position in the hierarchy of function is kept in mind
Daniel E Atkinson
Cellular Energy Metabolism and Its Regulation (1977)
KEY QUESTIONS
27.1 Can Systems Analysis Simplify the Complexity of Metabolism?
27.2 What Underlying Principle Relates ATP Coupling to the Thermodynamics of Metabolism?
27.3 Is There a Good Index of Cellular Energy Status?
27.4 How Is Overall Energy Balance Regulated
in Cells?
27.5 How Is Metabolism Integrated in
a Multicellular Organism?
27.6 What Regulates Our Eating Behavior?
27.7 Can You Really Live Longer by Eating Less?
ESSENTIAL QUESTIONS
Cells are systems in a dynamic steady state, maintained by a constant flux of
nutri-ents that serve as energy sources or as raw material for the maintenance of cellular
structures Catabolism and anabolism are ongoing, concomitant processes.
What principles underlie the integration of catabolism and energy production
with anabolism and energy consumption? How is metabolism integrated in
complex organisms with multiple organ systems?
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Trang 8840 Chapter 27 Metabolic Integration and Organ Specialization
macromolecular synthesis is driven by energy from ATP, although indirectly in some cases: GTP is the principal energy source for protein synthesis, CTP for phospholipid synthesis, and UTP for polysaccharide synthesis However, keep in mind that ATP is the principal phosphoryl donor for formation of GTP, CTP, and UTP from GDP, CDP, and UDP, respectively Macromolecules are the agents of biological function and information—proteins, nucleic acids, lipids that self-assemble into membranes, and
so on Growth can be represented as cellular accumulation of macromolecules and the partitioning of these materials of function and information into daughter cells in the process of cell division.
Only a Few Intermediates Interconnect the Major Metabolic Systems
Despite the complexity of processes taking place within each block, the connections between blocks involve only a limited number of substances Just ten or so kinds of catabolic intermediates from glycolysis, the pentose phosphate pathway, and the cit-ric acid cycle serve as the raw material for most of anabolism: four kinds of sugar phosphates (triose-P, tetrose-P, pentose-P, and hexose-P), three -keto acids
(pyru-vate, oxaloacetate, and -ketoglutarate), two coenzyme A derivatives (acetyl-CoA and
succinyl-CoA), and PEP (phosphoenolpyruvate)
ATP and NADPH Couple Anabolism and Catabolism
Metabolic intermediates are consumed by anabolic reactions and must be continu-ously replaced by catabolic processes In contrast, the energy-rich compounds ATP and NADPH are recycled rather than replaced When these substances are used in
Carbohydrates
CATABOLISM Fat
Protein
ANABOLISM
Triose-P Tetrose-P Pentose-P Hexose-P PEP Pyruvate Acetyl-CoA
-KG
Succinyl-CoA Oxaloacetate
Amino acids Nucleotides Fatty acids, etc
MACROMOLECULAR SYNTHESIS AND ACCUMULATION (GROWTH)
Proteins Nucleic acids Complex lipids
Membranes Organelles Cell walls, etc
+
FIXATION
h
ATP
ADP
NTP
NDP NADP+
NADPH
ATP
ADP
H2O
H2O
O2
CO2
CO2
NADP+
NADPH
FIGURE 27.1 Block diagram of intermediary metabolism
Trang 9biosynthesis, the products are ADP and NADP, and ATP and NADPH are
regener-ated during catabolism ATP and NADPH are unique in that they are the only
com-pounds whose purpose is to couple the energy-yielding processes of catabolism to the
energy-consuming reactions of anabolism Certainly, other coupling agents serve
es-sential roles in metabolism For example, NADH and [FADH2] participate in the
transfer of electrons from substrates to O2during oxidative phosphorylation
How-ever, these reactions are solely catabolic, and the functions of NADH and [FADH2]
are fulfilled within the block called catabolism
Phototrophs Have an Additional Metabolic System—
The Photochemical Apparatus
The systems in Figure 27.1 reviewed thus far are representative only of metabolism as
it exists in aerobic heterotrophs The photosynthetic production of ATP and NADPH
in photoautotrophic organisms entails a fourth block, the photochemical system
(Fig-ure 27.1) This block consumes H2O and releases O2 When this fourth block operates,
energy production within the catabolic block can be largely eliminated Yet another
block, one to account for the fixation of carbon dioxide into carbohydrates, is also
re-quired for photoautotrophs The inputs to this fifth block are the products of the
pho-tochemical system (ATP and NADPH) and CO2derived from the environment The
carbohydrate products of this block may enter catabolism, but not primarily for energy
production In photoautotrophs, carbohydrates are fed into catabolism to generate
the metabolic intermediates needed to supply the block of anabolism Although these
diagrams are oversimplifications of the total metabolic processes in heterotrophic or
phototrophic cells, they are useful illustrations of functional relationships between the
major metabolic subdivisions This general pattern provides an overall perspective on
metabolism, making its purpose easier to understand.
27.2 What Underlying Principle Relates ATP Coupling
to the Thermodynamics of Metabolism?
Virtually every metabolic pathway either consumes or produces ATP The amount of
ATP involved—that is, the stoichiometry of ATP synthesis or hydrolysis—lies at the
heart of metabolic relationships The overall thermodynamic efficiency of any
meta-bolic sequence, be it catameta-bolic or anameta-bolic, is determined by ATP coupling In every
case, the overall reaction mediated by any metabolic pathway is energetically favorable because of
its particular ATP stoichiometry In the highly exergonic reactions of catabolism, much of
the energy released is captured in ATP synthesis In turn, the thermodynamically
un-favorable reactions of anabolism are driven by energy released upon ATP hydrolysis
To illustrate this principle, we must first consider the three types of
stoichi-ometries The first two are fixed by the laws of chemistry, but the third is unique to
living systems and reveals a fundamental difference between the inanimate world of
chemistry and physics and the world of biological function, as shaped by evolution—
that is, the world of living organisms The fundamental difference is the
stoichiome-try of ATP coupling.
1 Reaction Stoichiometry This is simple chemical stoichiometry—the number of
each kind of atom in any chemical reaction remains the same, and thus equal
num-bers must be present on both sides of the equation This requirement holds even
for a process as complex as cellular respiration:
C6H12O6 6 O2⎯⎯→ 6 CO2 6 H2O The six carbons in glucose appear as 6 CO2, the 12 H of glucose appear as the 12 H
in six molecules of water, and the 18 oxygens are distributed between CO2and H2O.
2 Obligate Coupling Stoichiometry Cellular respiration is an oxidation–reduction
process, and the oxidation of glucose is coupled to the reduction of NADand [FAD].
(Brackets here denote that the relevant FAD is covalently linked to succinate
dehy-Stoichiometry is the measurement of the
amounts of chemical elements and molecules involved in chemical reactions (from the Greek
stoicheion, meaning “element,” and metria,
mean-ing “measure”)
Trang 10842 Chapter 27 Metabolic Integration and Organ Specialization
drogenase; see Chapter 20) The NADH and [FADH2] thus formed are oxidized in the electron-transport pathway:
(a) C6H12O6 10 NADⴙ 2 [FAD] 6 H2O ⎯⎯→
6 CO2 10 NADH 10 H 2 [FADH2] (b) 10 NADH 10 Hⴙ 2 [FADH2] 6 O2⎯⎯→ 12 H2O 10 NADⴙ 2 [FAD] Sequence (a) accounts for the oxidation of glucose via glycolysis and the citric acid cycle Sequence (b) is the overall equation for electron transport per glucose The
stoichiometry of coupling by the biological ecarriers, NADand FAD, is fixed by the
chemistry of electron transfer; each of the coenzymes serves as an epair acceptor
Re-duction of each O atom takes an epair Metabolism must obey these facts of
chem-istry: Biological oxidation of glucose releases 12 epairs, creating a requirement for
12 equivalents of epair acceptors, which transfer the electrons to 12 O atoms By evo-lutionary chance, NAD/NADH and FAD/FADH2carry these electrons, but the stoi-chiometry is fixed by the chemistry.
3 Evolved Coupling Stoichiometries The participation of ATP is fundamentally different from the role played by pyridine nucleotides and flavins The stoichiome-try of adenine nucleotides in metabolic sequences is not fixed by chemical neces-sity Instead, the “stoichiometries” we observe are the consequences of evolutionary design The overall equation for cellular respiration,1 including the coupled for-mation of ATP by oxidative phosphorylation, is
C6H12O6 6 O2 38 ADP 38 Pi⎯⎯→ 6 CO2 38 ATP 44 H2O The “stoichiometry” of ATP formation, 38 ADP 38 Pi⎯ →38 ATP 38 H2O, can-not be predicted from any chemical considerations The value of 38 ATP is an end result of biological adaptation It is a trait that evolved through interactions between chemistry, heredity, and the environment over the course of evolution Like any evolved character, ATP stoichiometry is the result of compromise The final trait is one particularly suited to the fitness of the organism.
The number 38 is not magical Recall that in eukaryotes, the consensus value for the net yield of ATP per glucose is 30 to 32, not 38 (see Table 20.4) Also, the value
of 38 was established a long time ago in evolution, when the prevailing atmospheric conditions and the competitive situation were undoubtedly very different from those today The significance of this number is that it provides a high yield of ATP for each glucose molecule, yet the yield is still low enough that essentially all of the glucose is metabolized.
ATP Coupling Stoichiometry Determines the Keqfor Metabolic Sequences
The fundamental biological purpose of ATP as an energy-coupling agent is to drive thermodynamically unfavorable reactions In effect, the energy release accompa-nying ATP hydrolysis is transmitted to the unfavorable reaction so that the overall free energy change for the coupled process is negative (that is, favorable) The in-volvement of ATP serves to alter the free energy change for a reaction; or to put it another way, the role of ATP is to change the equilibrium ratio of [reactants] to [products] for a reaction (See the A Deeper Look box on page 67.)
Another way of viewing these relationships is to note that, at equilibrium, the concentrations of ADP and Piwill be vastly greater than that of ATP because G°
for ATP hydrolysis is a large negative number.2However, the cell where this reaction
1This overall equation for cellular respiration is for the reaction within an uncompartmentalized (prokaryotic) cell In eukaryotes, where much of the cellular respiration is compartmentalized within mitochondria, mitochondrial ADP/ATP exchange imposes a metabolic cost on the proton gradient
of 1 Hper ATP, so the overall yield of ATP per glucose is 32, not 38
2Since G° 30.5 kJ/mol, ln Keq 12.3 So Keq 2.2 105 Choosing starting conditions of [ATP] 8 mM, [ADP] 8 mM, and [Pi] 1 mM, we can assume that, at equilibrium, [ATP] has fallen to some insignificant value x, [ADP] approximately 16 mM, and [Pi] approximately 9 mM The concentration of ATP at equilibrium, x, then calculates to be about 1 nM.