Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

In organic solvents N C

In lipid membrane

C

polar residues (making these complexes freely soluble in the hydrophobic membrane interior).

PROBLEMS

1.Calculate the free energy difference at 25°C due to a galactose gradient across a membrane, if the concentration on side 1 is 2 mM and the concentration on side 2 is 10 mM.

2.Consider a phospholipid vesicle containing 10 mM Na ions. The vesicle is bathed in a solution that contains 52 mM Na ions,

and the electrical potential difference across the vesicle mem-

brane outside inside 30 mV. What is the electrochemical potential at 25°C for Na ions?

3. Transport of histidine across a cell membrane was measured at several histidine concentrations:

Does this transport operate by passive diffusion or by facilitated diffusion?

FURTHER READING

Benz, R., 1980. Structure and function of porins from Gram-negative bacteria. Annual Review of Microbiology 42:359–393.

Blair, H. C., et al., 1989. Osteoclastic bone resorption by a polarized vacuolar proton pump. Science 245:855–857.

Christensen, B., et al., 1988. Channel-forming properties of cecropins and related model compounds incorporated into planar lipid membranes. Proceedings of the National Academy of Sciences U.S.A. 85:5072–5076.

Choe, S., Bennett, M., Fujii, G., et al., 1992. The crystal structure of diphtheria toxin. Nature 357:216–222.

Collarini, E. J., and Oxender, D., 1987. Mechanisms of transport of amino acids across membrane. Annual Review of Nutrition 7:75–90.

Featherstone, C., 1990. An ATP-driven pump for secretion of yeast mating factor. Trends in Biochemical Sciences 15:169–170.

Garavito, R. M., et al., 1983. X-ray diffraction analysis of matrix porin, an integral membrane protein from Escherichia coli outer membrane.

Journal of Molecular Biology 164:313–327.

Glynn, I., and Karlish, S., 1990. Occluded ions in active transport. Annual Review of Biochemistry 59:171–205.

Gould, G. W., and Bell, G. I., 1990. Facilitative glucose transporters: An expanding family. Trends in Biochemical Sciences 15:18–23.

Hughson, F., 1997. Penetrating insights into pore formation. Nature Structural Biology 4:89–92.

Inesi, G., Sumbilla, C., and Kirtley, M., 1990. Relationships of molecular structure and function in Ca2 -transport ATPase. Physiological Reviews 70:749–759.

4. Fructose is present outside a cell at 1 M concentration. An active transport system in the plasma membrane transports fructose into this cell, using the free energy of ATP hydrolysis to drive fructose uptake. Assume that one fructose is transported per ATP hydrolyzed, that ATP is hydrolyzed on the intracellular surface of the membrane, and that the concentrations of ATP, ADP, and Pi are 3 mM, 1 mM, and 0.5 mM, respectively. T 298 K. What is the highest intracellular concentration of fructose that this transport system can generate? (Hint: Refer to Chapter 3 to recall the effects of concentration on free energy of ATP hydrolysis.)

5.The rate of K transport across bilayer membranes reconstituted from dipalmitoylphosphatidylcholine (DPPC) and nigericin is approximately the same as that observed across membranes reconstituted from DPPC and cecropin a at 35°C. Would you expect the transport rates across these two membranes also to be similar at 50°C? Explain.

6.In this chapter, we have examined coupled transport systems that rely on ATP hydrolysis, on primary gradients of Na or H ,

and on phosphotransferase systems. Suppose you have just discovered an unusual strain of bacteria that transports rhamnose across its plasma membrane. Suggest experiments that would test whether it was linked to any of these other transport systems.

Jap, B., and Walian, P. J., 1990. Biophysics of the structure and function of porins. Quarterly Reviews of Biophysics 23:367–403.

Jay, D., and Cantley, L., 1986. Structural aspects of the red cell anion exchange protein. Annual Review of Biochemistry 55:511–538.

Jennings, M. L., 1989. Structure and function of the red blood cell anion transport protein. Annual Review of Biophysics and Biophysical Chemistry

18:397–430.

Jørgensen, P. L., 1986. Structure, function and regulation of Na ,K - ATPase in the kidney. Kidney International 29:10–20.

Jørgensen, P. L., and Andersen, J. P., 1988. Structural basis for E1-E2 conformational transitions in Na ,K -pump and Ca2 -pump proteins.

Journal of Membrane Biology 103:95–120.

Juranka, P. F., Zastawny, R. L., and Ling, V., 1989. P-Glycoprotein: Multidrug-resistance and a superfamily of membrane-associated transport proteins. The FASEB Journal 3:2583–2592.

Kaback, H. R., Bibi, E., and Roepe, P. D., 1990. -Galactoside transport in E. coli: A functional dissection of lac permease. Trends in Biochemical Sciences 15:309–314.

Kartner, N., and Ling, V., 1989. Multidrug resistance in cancer. Scientific American 260:44–51.

Li, H., Lee, S., and Jap, B., 1997. Molecular design of aquaporin-1 water channel as revealed by electron crystallography. Nature Structural Biology 4:263–265.

Li, J., Carroll, J., and Ellar, D., 1991. Crystal structure of insecticidal -endo- toxin from Bacillus thuringiensis at 2.5 Å resolution. Nature 353:815–821.

326 Chapter 10 ● Membrane Transport

Matthew, M. K., and Balaram, A., 1983. A helix dipole model for alamethicin and related transmembrane channels. FEBS Letters 157:1–5.

Meadow, N. D., Fox, D. K., and Roseman, S., 1990. The bacterial phosphoenolpyruvate:glycose phosphotransferase system. Annual Review of Biochemistry 59:497–542.

Miller, C., 1996. A chloride channel model? Science 274:738.

Oesterhelt, D., and Tittor, J., 1989. Two pumps, one principle: Lightdriven ion transport in Halobacteria. Trends in Biochemical Sciences 14:57–61.

Parker, M., 1997. More than one way to make a hole. Nature Structural Biology 4:250–253.

Parker, M., Buckley, J., Postma, J., et al., 1994. Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states. Nature 367:292–295.

Parker, M., Tucker, A., Tsernoglou, D., and Pattus, F., 1990. Insights into membrane insertion based on studies of colicins. Trends in Biochemical Sciences 15:126–129.

Pedersen, P. L., and Carafoli, E., 1987. Ion motive ATPases. Trends in Biochemical Sciences 12:146–150, 186–189.

Petosa, C., Collier, R., Klimpel, K., et al., 1997. Crystal structure of the anthrax toxin protective antigen. Nature 385:833–838.

Pressman, B., 1976. Biological applications of ionophores. Annual Review of Biochemistry 45:501–530.

Prince, R. C., 1990. At least one Bacillus thuringiensis toxin forms ionselective pores in membranes. Trends in Biochemical Sciences 15:2–3.

Prince, R., Gunson, D., and Scarpa, A., 1985. Sting like a bee! The ionophoric properties of melittin. Trends in Biochemical Sciences 10:99.

Schirmer, T., Keller, T. A., Wang, Y.-F., and Rosenbusch, J. P., 1995. Structural basis for sugar translocation through maltoporin channels at 3.1 Å resolution. Science 267:512–514.

Song, L., Hobaugh, M., Shustak, C., et al., 1996. Structure of staphylococcal -hemolysin, a heptameric transmembrane pore. Science 274:1859 –1866.

Spudich, J. L., and Bogomolni, R. A., 1988. Sensory rhodopsins of

Halobacteria. Annual Review of Biophysics and Biophysical Chemistry 17:193– 215.

Wade, D., et al., 1990. All-D amino acid-containing channel-forming antibiotic peptides. Proceedings of the National Academy of Sciences U.S.A.

87:4761–4765.

Wallace, B. A., 1990. Gramicidin channels and pores. Annual Review of Biophysics and Biophysical Chemistry 19:127–157.

Walmsley, A. R., 1988. The dynamics of the glucose transporter. Trends in Biochemical Sciences 13:226–231.

Weiner, M., Freymann, D., Ghosh, P., and Stroud, R., 1997. Crystal structure of colicin Ia. Nature 385:461–464.

Wheeler, T. J., and Hinkle, P., 1985. The glucose transporter of mammalian cells. Annual Review of Physiology 47:503–517.

Chapter 11

Nucleotides and cleic Acids

Francis Crick and James Watson point out features of their model for the

structure of DNA. (© A. Barrington Brown/Science Source/Photo Researchers, Inc.)

Nucleotides and nucleic acids are biological molecules that possess heterocyclic nitrogenous bases as principal components of their structure. The biochemical roles of nucleotides are numerous; they participate as essential intermediates in virtually all aspects of cellular metabolism. Serving an even more central biological purpose are the nucleic acids, the elements of heredity and the agents of genetic information transfer. Just as proteins are linear polymers of amino acids, nucleic acids are linear polymers of nucleotides. Like the letters in this sentence, the orderly sequence of nucleotide residues in a nucleic acid can encode information. The two basic kinds of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Complete hydrolysis of nucleic acids liberates nitrogenous bases, a five-carbon sugar, and phosphoric acid in equal amounts. The five-carbon sugar in DNA is 2-deoxyribose; in RNA,

We have discovered the secret of life!

Proclamation by Francis H. C. Crick to patrons of The Eagle, a pub in Cambridge, England (1953)

OUTLINE

11.1 ● Nitrogenous Bases

11.2 ● The Pentoses of Nucleotides and

Nucleic Acids

11.3 ● Nucleosides Are Formed by Joining a Nitrogenous Base to a Sugar

11.4 ● Nucleotides Are Nucleoside Phosphates

11.5 ● Nucleic Acids Are Polynucleotides

11.6 ● Classes of Nucleic Acids

11.7 ● Hydrolysis of Nucleic Acids

327

328 Chapter 11 ● Nucleotides and Nucleic Acids

DNA

Replication

Transcription

DNA

mRNA

tRNAs

Ribosome

Attached

amino acid Growing

peptide chain

Replication

DNA replication yields two DNA molecules identical to the

original one, ensuring transmission of genetic information to daughter cells with exceptional fidelity.

Transcription

The sequence of bases in DNA is recorded as a sequence of complementary bases in a singlestranded mRNA molecule.

Translation

Three-base codons on the mRNA corresponding to specific amino acids direct the sequence of building a protein. These codons are recognized by tRNAs (transfer RNAs) carrying the appropriate amino acids. Ribosomes are the “machinery” for protein synthesis.

FIGURE 11.2 ● (a) The pyrimidine ring system; by convention, atoms are numbered as indicated. (b) The purine ring system, atoms numbered as shown.

it is ribose. (See Chapter 7 for a detailed discussion of sugars and other carbohydrates.) DNA is the repository of genetic information in cells, while RNA serves in the transcription and translation of this information (Figure 11.1). An interesting exception to this rule is that some viruses have their genetic information stored as RNA.

This chapter describes the chemistry of nucleotides and the major classes of nucleic acids. Chapter 12 presents methods for determination of nucleic acid primary structure (nucleic acid sequencing) and describes the higher orders of nucleic acid structure. Chapter 13 introduces the molecular biology of recombinant DNA: the construction and uses of novel DNA molecules assembled by combining segments from other DNA molecules.

11.1 ● Nitrogenous Bases

The bases of nucleotides and nucleic acids are derivatives of either pyrimidine or purine. Pyrimidines are six-membered heterocyclic aromatic rings containing two nitrogen atoms (Figure 11.2a). The atoms are numbered in a clockwise fashion, as shown in the figure. The purine ring structure is represented by the combination of a pyrimidine ring with a five-membered imidazole ring to yield a fused ring system (Figure 11.2b). The nine atoms in this system are numbered according to the convention shown.

The pyrimidine ring system is planar, while the purine system deviates somewhat from planarity in having a slight pucker between its imidazole and pyrimidine portions. Both are relatively insoluble in water, as might be expected from their pronounced aromatic character.

Common Pyrimidines and Purines

The common naturally occurring pyrimidines are cytosine, uracil, and thymine (5-methyluracil) (Figure 11.3). Cytosine and thymine are the pyrimidines typically found in DNA, whereas cytosine and uracil are common in RNA. To view this generality another way, the uracil component of DNA occurs as the 5- methyl variety, thymine. Various pyrimidine derivatives, such as dihydrouracil, are present as minor constituents in certain RNA molecules.

Adenine (6-amino purine) and guanine (2-amino-6-oxy purine), the two common purines, are found in both DNA and RNA (Figure 11.4). Other naturally occurring purine derivatives include hypoxanthine, xanthine, and uric acid (Figure 11.5). Hypoxanthine and xanthine are found only rarely as constituents of nucleic acids. Uric acid, the most oxidized state for a purine derivative, is never found in nucleic acids.

● The common purine bases—adenine and guanine—in the tautomeric forms predominant at pH 7.

Properties of Pyrimidines and Purines

The aromaticity of the pyrimidine and purine ring systems and the electronrich nature of their OOH and ONH2 substituents endow them with the capacity to undergo keto–enol tautomeric shifts. That is, pyrimidines and purines exist as tautomeric pairs, as shown in Figure 11.6 for uracil. The keto tautomer is called a lactam, whereas the enol form is a lactim. The lactam form vastly predominates at neutral pH. In other words, pKa values for ring nitrogen atoms 1 and 3 in uracil are greater than 8 (the pKa value for N-3 is 9.5) (Table 11.1).

Table 11.1

Proton Dissociation Constants (pKa Values) for Nucleotides

Thymine (2-oxy-4-oxy 5-methyl pyrimidine)

● The common pyrimidine bases—cytosine, uracil, and thymine—in the tautomeric forms predominant at pH 7.

O

H

N N

NN H

Hypoxanthine

O

H

N N

O N N

H H

Xanthine

O H

H

N N

O

O N N

H H

Uric acid

FIGURE 11.5 ● Other naturally occurring purine derivatives—hypoxanthine, xanthine, and uric acid.

FIGURE 11.6 ● The keto/enol tautomerism of uracil.

329

Absorbance

5'-AMP

1.0

pH 7

0.8

0.6

0.4

pH 2

0.2

0

220 240 260 280 300 Wavelength, nm

11.2 ● The Pentoses of Nucleotides and Nucleic Acids

Five-carbon sugars are called pentoses (see Chapter 7). RNA contains the pentose D-ribose, while 2-deoxy-D-ribose is found in DNA. In both instances, the pentose is in the five-membered ring form known as furanose: D-ribofuranose for RNA and 2-deoxy-D-ribofuranose for DNA (Figure 11.9). When these ribofuranoses are found in nucleotides, their atoms are numbered as 1 , 2 , 3 , and so on to distinguish them from the ring atoms of the nitrogenous bases. As we shall see, the seemingly minor difference of a hydroxyl group at the 2 - position has far-reaching effects on the secondary structures available to RNA and DNA, as well as their relative susceptibilities to chemical and enzymatic hydrolysis.

FIGURE 11.8 ● The UV absorption spectra of the common ribonucleotides.