Berg J.M., Tymoczko J.L., Stryer L. Biochemistry
Подождите немного. Документ загружается.
9.
Inhibitor design. Compound A has been synthesized as a potential inhibitor of a urea-cycle enzyme. Which enzyme
do you think compound A might inhibit?
See answer
10.
Ammonia toxicity. Glutamate is an important neurotransmitter whose levels must be carefully regulated in the brain.
Explain how a high concentration of ammonia might disrupt this regulation. How might a high concentration of
ammonia alter the citric acid cycle?
See answer
11.
A precise diagnosis. The urine of an infant gives a positive reaction with 2,4-dinitrophenylhydrazine. Mass
spectrometry shows abnormally high blood levels of pyruvate, α-ketoglutarate, and the α-ketoacids of valine,
isoleucine, and leucine. Identify a likely molecular defect and propose a definitive test of your diagnosis.
See answer
12.
Therapeutic design. How would you treat an infant who is deficient in argininosuccinate synthetase? Which
molecules would carry nitrogen out of the body?
See answer
13.
Sweet hazard. Why should phenylketonurics avoid using aspartame, an artificial sweetener? (Hint: Aspartame is l-
aspartyl-l-phenylalanine methyl ester.)
See answer
14.
Déjà vu. N-Acetylglutamate is required as a cofactor in the synthesis of carbamoyl phosphate. How might N-
acetylglutamate be synthesized from glutamate?
See answer
Mechanism Problems
15.
Serine dehydratase. Write out a complete mechanism for the conversion of serine into aminoacrylate catalyzed by
serine dehydratase.
See answer
16.
Serine racemase. The nervous system contains a substantial amount of d-serine, which is generated from l-serine by
serine racemase, a PLP-dependent enzyme. Propose a mechanism for this reaction. What is the equilibrium constant
for the reaction l-serine d-serine?
See answer
Chapter Integration Problems
17.
Double duty. Degradation signals are commonly located in protein regions that also facilitate protein-protein
interactions. Explain why this coexistence of two functions in the same domain might be useful.
See answer
18.
Fuel choice. Within a few days after a fast begins, nitrogen excretion accelerates to a higher-than-normal level.
After a few weeks, the rate of nitrogen excretion falls to a lower level and continues at this low rate. However, after
the fat stores have been depleted, nitrogen excretion rises to a high level.
(a) What events trigger the initial surge of nitrogen excretion?
(b) Why does nitrogen excretion fall after several weeks of fasting?
(c) Explain the increase in nitrogen excretion when the lipid stores have been depleted.
See answer
19.
Isoleucine degradation. Isoleucine is degraded to acetyl CoA and succinyl CoA. Suggest a plausible reaction
sequence, based on reactions discussed in the text, for this degradation pathway.
See answer
Data Interpretation Problem
20.
Another helping hand. In eukaryotes, the 20S proteasome in conjunction with the 19S component degrades
ubiquitinated proteins with the hydrolysis of a molecule of ATP. Archaea lack ubiquitin and the 26S proteasome
but do contain a 20S proteasome. Some archaea also contain an ATPase that is homologous to the ATPases of the
eukaryotic 19S component. This archaeal ATPase activity was isolated as a 650-kd complex (called PAN) from the
archaeon Thermoplasma, and experiments were performed to determine if PAN could enhance the activity of the
20S proteasome from Thermoplasma as well as other 20S proteasomes.
Protein degradation was measured as a function of time and in the presence of various combinations of
components. Graph A shows the results.
(a) What is the effect of PAN on archaeal proteasome activity in the absence of nucleotides?
(b) What is the nucleotide requirement for protein digestion?
(c) What evidence suggests that ATP hydrolysis, and not just the presence of ATP, is required for digestion?
A similar experiment was performed with a small peptide as a substrate for the proteasome instead of a protein. The
results obtained are shown in graph B.
(d) How do the requirements for peptide digestion differ from those of protein digestion?
(e) Suggest some reasons for the difference.
The ability of PAN from the archaeon Thermoplasma to support protein degradation by the 20S proteasomes from
the archeaon Methanosarcina and rabbit muscle was then examined.
(f) Can the Thermoplasma PAN augment protein digestion by the proteasomes from other organisms?
(g) What is the significance of the stimulation of rabbit muscle proteasome by Thermoplasma PAN?
[Data from P. Zwickl, D. Ng, K. M. Woo, H.-P. Klenk, and A. L. Goldberg. An archaebacterial ATPase,
homologous to ATPase in the eukaryotic 26S proteasome, activates protein breakdown by 20S proteasomes. J.
Biol. Chem. 274(1999): 26008
26014.]
See answer
II. Transducing and Storing Energy 23. Protein Turnover and Amino Acid Catabolism
Selected Readings
Where to start
Y.M. Torchinsky. 1989. Transamination: Its discovery, biological and chemical aspects Trends Biochem. Sci. 12: 115-
117. (PubMed)
R.C. Eisensmith and S.L.C. Woo. 1991. Phenylketonuria and the phenylalanine hydroxylase gene Mol. Biol. Med. 8: 3-
18. (PubMed)
H.L. Levy. 1989. Nutritional therapy for selected inborn errors of metabolism J. Am. Coll. Nutr. 8: 54S-60S. (PubMed)
A.L. Schwartz and A. Ciechanover. 1999. The ubiquitin-proteasome pathway and pathogenesis of human diseases Annu.
Rev. Med. 50: 57-74. (PubMed)
Books
Bender, D. A., 1985. Amino Acid Metabolism (2d ed.). Wiley.
Lippard, S. J., and Berg, J. M., 1994. Principles of Bioinorganic Chemistry. University Science Books.
Schauder, P., Wahren, J., Paoletti, R., Bernardi, R., and Rinetti, M. (Eds.), 1992. Branched-Chain Amino Acids:
Biochemistry, Physiopathology, and Clinical Sciences. Raven Press.
Grisolia, S., Báguena, R., and Mayor, F. (Eds.), 1976. The Urea Cycle. Wiley.
Walsh, C., 1979. Enzymatic Reaction Mechanisms. W. H. Freeman and Company.
Christen, P., and Metzler, D. E., 1985. Transaminases. Wiley.
Ubiquitin and the proteasome
M. Bochtler, L. Ditzel, M. Groll, C. Hartmann, and R. Huber. 1999. The proteasome Annu. Rev. Biophys. Biomol. Struct.
28: 295-317. (PubMed)
J.S. Thrower, L. Hoffman, M. Rechsteiner, and C.M. Pickart. 2000. Recognition of the polyubiquitin proteolytic signal
EMBO J. 19: 94-102. (PubMed)
M. Hochstrasser. 2000. Evolution and function of ubiquitin-like protein-conjugation systems Nat. Cell Biol. 2: E153-
E157. (PubMed)
S. Jentsch and G. Pyrowolakis. 2000. Ubiquitin and its kin: How close are the family ties? Trends Cell Biol. 10: 335-342.
(PubMed)
J.D. Laney and M. Hochstrasser. 1999. Substrate targeting in the ubiquitin system Cell 97: 427-430. (PubMed)
W.J. Cook, L.C. Jeffrey, E. Kasperek, and C.M. Pickart. 1994. Structure of tetraubiquitin shows how multiubiquitin
chains can be formed J. Mol. Biol. 236: 601-609. (PubMed)
R. Hartmann-Petersen, K. Tanaka, and K.B. Hendil. 2001. Quaternary structure of the ATPase complex of human 26S
proteasomes determined by chemical cross-linking Arch. Biochem. Biophys. 386: 89-94. (PubMed)
Pyridoxal phosphate-dependent enzymes
P.K. Mehta and P. Christen. 2000. The molecular evolution of pyridoxal-5
-phosphate-dependent enzymes Adv.
Enzymol. Relat. Areas Mol. Biol. 74: 129-184. (PubMed)
G. Schneider, H. Kack, and Y. Lindqvist. 2000. The manifold of vitamin B
6
dependent enzymes Structure Fold Des. 8:
R1-R6. (PubMed)
J. Jager, M. Moser, U. Sauder, and J.N. Jansonius. 1994. Crystal structures of Escherichia coli aspartate
aminotransferase in two conformations: Comparison of an unliganded open and two liganded closed forms J. Mol. Biol.
239: 285-305. (PubMed)
V.N. Malashkevich, M.D. Toney, and J.N. Jansonius. 1993. Crystal structures of true enzymatic reaction intermediates:
Aspartate and glutamate ketimines in aspartate aminotransferase Biochemistry 32: 13451-13462. (PubMed)
C.A. McPhalen, M.G. Vincent, D. Picot, J.N. Jansonius, A.M. Lesk, and C. Chothia. 1992. Domain closure in
mitochondrial aspartate aminotransferases J. Mol. Biol. 227: 197-213. (PubMed)
Urea-cycle enzymes
X. Huang and F.M. Raushel. 2000. Restricted passage of reaction intermediates through the ammonia tunnel of
carbamoyl phosphate synthetase J. Biol. Chem. 275: 26233-26240. (PubMed)
F.S. Lawson, R.L. Charlebois, and J.A. Dillon. 1996. Phylogenetic analysis of carbamoylphosphate synthetase genes:
Complex evolutionary history includes an internal duplication within a gene which can root the tree of life Mol. Biol.
Evol. 13: 970-977. (PubMed)
C.R. McCudden and S.G. Powers-Lee. 1996. Required allosteric effector site for N-acetylglutamate on carbamoyl-
phosphate synthetase I J. Biol. Chem. 271: 18285-18294. (PubMed)
Z.F. Kanyo, L.R. Scolnick, D.E. Ash, and D.W. Christianson. 1996. Structure of a unique binuclear manganese cluster in
arginase Nature 383: 554-557. (PubMed)
M.A. Turner, A. Simpson, R.R. McInnes, and P.L. Howell. 1997. Human argininosuccinate lyase: A structural basis for
intragenic complementation Proc. Natl. Acad. Sci. USA 94: 9063-9068. (PubMed) (Full Text in PMC)
Amino acid degradation
F. Fusetti, H. Erlandsen, T. Flatmark, and R.C. Stevens. 1998. Structure of tetrameric human phenylalanine hydroxylase
and its implications for phenylketonuria J. Biol. Chem. 273: 16962-16967. (PubMed)
K. Sugimoto, T. Senda, H. Aoshima, E. Masai, M. Fukuda, and Y. Mitsui. 1999. Crystal structure of an aromatic ring
opening dioxygenase LigAB, a protocatechuate 4,5-dioxygenase, under aerobic conditions Structure Fold Des. 7: 953-
965. (PubMed)
G.P. Titus, H.A. Mueller, J. Burgner, S. Rodriguez De Cordoba, M.A. Penalva, and D.E. Timm. 2000. Crystal structure
of human homogentisate dioxygenase Nat. Struct. Biol. 7: 542-546. (PubMed)
H. Erlandsen and R.C. Stevens. 1999. The structural basis of phenylketonuria Mol. Genet. Metab. 68: 103-125.
(PubMed)
Genetic diseases
Striver, C. R., Beaudet, A. L., Sly, W. S., Valle, D., Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S. (Eds.),
1995. The Metabolic Basis of Inherited Diseases (7th ed.). McGraw-Hill.
Nyhan, W. L. (Ed.), 1984. Abnormalities in Amino Acid Metabolism in Clinical Medicine. Appleton-Century-Crofts.
Historical aspects and the process of discovery
A.J.L. Cooper and A. Meister. 1989. An appreciation of Professor Alexander E. Braunstein: The discovery and scope of
enzymatic transamination Biochimie 71: 387-404. (PubMed)
Garrod, A. E., 1909. Inborn Errors in Metabolism. Oxford University Press (reprinted in 1963 with a supplement by H.
Harris).
B. Childs. 1970. Sir Archibald Garrod's conception of chemical individuality: A modern appreciation N. Engl. J. Med.
282: 71-78. (PubMed)
F.L. Holmes. 1980. Hans Krebs and the discovery of the ornithine cycle Fed. Proc. 39: 216-225. (PubMed)
III. Synthesizing the Molecules of Life
Nitrogenase complex. Nitrogen is an essential component of many biochemical building blocks. The enzyme complex
shown here converts nitrogen gas, an abundant but inert compound, into a form that can be used for synthesizing amino
acids, nucleotides, and other biochemicals.
III. Synthesizing the Molecules of Life
24. The Biosynthesis of Amino Acids
The assembly of biological molecules, including proteins and nucleic acids, requires the generation of appropriate
starting materials. We have already considered the assembly of carbohydrates in regard to the Calvin cycle and the
pentose phosphate pathway (Chapter 20). The present chapter and the next two examine the assembly of the other
important building blocks namely, amino acids, nucleotides, and lipids.
The pathways for the biosynthesis of these molecules are extremely ancient, going back to the last common ancestor of
all living things. Indeed, these pathways probably predate many of the pathways of energy transduction discussed in Part
II and may have provided key selective advantages in early evolution. Many of the intermediates present in energy-
transduction pathways play a role in biosynthesis as well. These common intermediates allow efficient interplay between
energy-transduction (catabolic) and biosynthetic (anabolic) pathways. Thus, cells are able to balance the degradation of
compounds for energy mobilization and the synthesis of starting materials for macro-molecular construction.
We begin our consideration of biosynthesis with amino acids. Amino acids are the building blocks of proteins and the
nitrogen source for many other important molecules, including nucleotides, neurotransmitters, and prosthetic groups
such as porphyrins.
24.0.1. An Overview of Amino Acid Synthesis
A fundamental problem for biological systems is to obtain nitrogen in an easily usable form. This problem is solved by
certain microorganisms capable of reducing the inert N
N molecule (nitrogen gas) to two molecules of ammonia in one
of the most remarkable reactions in biochemistry. Nitrogen in the form of ammonia is the source of nitrogen for all the
amino acids. The carbon backbones come from the glycolytic pathway, the pentose phosphate pathway, or the citric acid
cycle.
Anabolism
Biosynthetic processes.
Degradative processes.
Derived from the Greek ana, "up"; kata, "down"; ballein, "to throw."
In amino acid production, we encounter an important problem in biosynthesis
namely, stereochemical control. Because
all amino acids except glycine are chiral, biosynthetic pathways must generate the correct isomer with high fidelity. In
each of the 19 pathways for the generation of chiral amino acids, the stereochemistry at the α-carbon atom is established
by a transamination reaction that involves pyridoxal phosphate. Almost all the transaminases that catalyze these
reactions descend from a common ancestor, illustrating once again that effective solutions to biochemical problems are
retained throughout evolution (Figure 24.1).
Biosynthetic pathways are often highly regulated such that building blocks are synthesized only when supplies are low.
Very often, a high concentration of the final product of a pathway inhibits the activity of enzymes that function early in
the pathway. Often present are allosteric enzymes capable of sensing and responding to concentrations of regulatory
species. These enzymes are similar in functional properties to aspartate transcarbamylase and its regulators (Section
10.1). Feedback and allosteric mechanisms ensure that all twenty amino acids are maintained in sufficient amounts for
protein synthesis and other processes.
III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids
Figure 24.1. Aminotransferase Protein Family. A superposition of the structures of four aminotransferases taking part
in amino acid biosynthesis reveals a common fold, indicating that these proteins descended from a common
ancestor.
III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids
Nitrogen is a key component of amino acids. The atmosphere is rich in nitrogen gas (N
2
), a very unreactive molecule.
Certain organisms such as bacteria that live in the root nodules of yellow clover (photo at left) can convert nitrogen gas
into ammonia. Ammonia can then be used to synthesize first glutamate and then other amino acids. [(Left) Runk/
Schoenberger from Grant Heilman]
III. Synthesizing the Molecules of Life 24. The Biosynthesis of Amino Acids
24.1. Nitrogen Fixation: Microorganisms Use ATP and a Powerful Reductant to
Reduce Atmospheric Nitrogen to Ammonia
The nitrogen in amino acids, purines, pyrimidines, and other biomolecules ultimately comes from atmospheric nitrogen,
N
2.
The biosynthetic process starts with the reduction of N
2
to NH
3
(ammonia), a process called nitrogen fixation.
Although higher organisms are unable to fix nitrogen, this conversion is carried out by some bacteria and archaea.
Symbiotic Rhizobium bacteria invade the roots of leguminous plants and form root nodules in which they fix nitrogen,
supplying both the bacteria and the plants. The amount of N
2
fixed by diazotrophic (nitrogen-fixing) microorganisms has
been estimated to be 10
11
kilograms per year, about 60% of Earth's newly fixed nitrogen. Lightning and ultraviolet
radiation fix another 15%; the other 25% is fixed by industrial processes. The industrial process for nitrogen fixation
devised by Fritz Haber in 1910 is still being used in fertilizer factories.
The fixation of N
2
is typically carried out by mixing with H
2
gas over an iron catalyst at about 500°C and a pressure of
300 atmospheres. The extremely strong N
N bond, which has a bond energy of 225 kcal mol
-1
, is highly resistant to
chemical attack. Indeed, Lavoisier named nitrogen gas "azote," meaning "without life" because it is so unreactive.
Nevertheless, the conversion of nitrogen and hydrogen to form ammonia is thermodynamically favorable; the reaction is
difficult kinetically because intermediates along the reaction pathway are unstable.
To meet the kinetic challenge, the biological process of nitrogen fixation requires a complex enzyme with multiple redox
centers. The nitrogenase complex, which carries out this fundamental transformation, consists of two proteins: a
reductase, which provides electrons with high reducing power, and nitrogenase, which uses these electrons to reduce N
2
to NH
3.
The transfer of electrons from the reductase to the nitrogenase component is coupled to the hydrolysis of ATP
by the reductase (Figure 24.2). The nitrogenase complex is exquisitely sensitive to inactivation by O
2
. Leguminous
plants maintain a very low concentration of free O
2
in their root nodules by binding O
2
to leghemoglobin, a homolog of
hemoglobin (Section 7.3.1).
In principle, the reduction of N
2
to NH
3
is a six-electron process.
However, the biological reaction always generates at least 1 mol of H
2
in addition to 2 mol of NH
3
for each mole of
N
N. Hence, an input of two additional electrons is required.
In most nitrogen-fixing microorganisms, the eight high-potential electrons come from reduced ferredoxin, generated by
photosynthesis or oxidative processes. Two molecules of ATP are hydrolyzed for each electron transferred. Thus, at
least 16 molecules of ATP are hydrolyzed for each molecule of N
2
reduced.
Again, ATP hydrolysis is not required to make nitrogen reduction favorable thermodynamically. Rather, it is essential to
reduce the heights of activation barriers along the reaction pathway, thus making the reaction kinetically feasible.
24.1.1. The Iron-Molybdenum Cofactor of Nitrogenase Binds and Reduces
Atmospheric Nitrogen
Both the reductase and the nitrogenase components of the complex are iron-sulfur proteins, in which iron is bonded to
the sulfur atom of a cysteine residue and to inorganic sulfide. The reductase (also called the iron protein or the Fe
protein) is a dimer of identical 30-kd subunits bridged by a 4Fe-4S cluster (Figure 24.3).
The role of the reductase is to transfer electrons from a suitable donor, such as reduced ferredoxin, to the
nitrogenase component. The binding and hydrolysis of ATP triggers a conformational change that moves the
reductase closer to the nitrogenase component from whence it is able to transfer its electron to the center of nitrogen
reduction. The structure of the ATP-binding region reveals it to be a member of the P-loop NTPase family (Section
9.4.1) that is clearly related to the nucleotide-binding regions found in G proteins and related proteins (Section 15.1.2).