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

Smooth Muscle Effectors Are Useful Drugs

The action of epinephrine and related agents forms the basis of therapeutic control of smooth muscle contraction. Breathing disorders, including asthma and various allergies, can result from excessive contraction of bronchial smooth muscle tissue. Treatment with epinephrine, whether by tablets or aerosol inhalation, inhibits MLCK and relaxes bronchial muscle tissue. More specific bronchodilators, such as albuterol (see figure), act more selec-

tively on the lungs and avoid the undesirable side effects of epinephrine on the heart. Albuterol is also used to prevent premature labor in pregnant women, owing to its relaxing effect on uterine smooth muscle. Conversely, oxytocin, known also as pitocin, stimulates contraction of uterine smooth muscle. This natural secretion of the pituitary gland is often administered to induce labor.

Filament

Hook

Periplasmic space

Plasma membrane

Cytosol

M ring

H+

Mot A

H+ Mot

B

i o

(c)

Restoring force, f

562

● A model of the flagellar motor assembly of Escherichia coli. The M ring carries an array of about 100 motB proteins at its periphery. These juxtapose with motA proteins in the protein complex that surrounds the ring assembly. Motion of protons through the motA/motB complexes drives the rotation of the rings and the associated rod and helical filament.

The rotations of bacterial flagellar filaments are the result of the rotation of motor protein complexes in the bacterial plasma membrane. The flagellar motor consists of at least two rings (including the M ring and the S ring) with diameters of about 25 nm assembled around and connected rigidly to a rod attached in turn to the helical filament (Figure 17.33). The rings are surrounded by a circular array of membrane proteins. In all, at least 40 genes appear to code for proteins involved in this magnificent assembly. One of these, the motB protein, lies on the edge of the M ring, where it interacts with the motA protein, located in the membrane protein array and facing the M ring.

In contrast to the many other motor proteins described in this chapter, a proton gradient, not ATP hydrolysis, drives the flagellar motor. The concentration of protons, [H ], outside the cell is typically higher than that inside the cell. Thus, there is a thermodynamic tendency for protons to move into the cell. The motA and motB proteins together form a proton shuttling device that is coupled to motion of the motor disks. Proton movement into the cell through this protein complex or “channel” drives the rotation of the flagellar motor. A model for this coupling has been proposed by Howard Berg and his coworkers (Figure 17.34). In this model, the motB proteins possess proton exchanging sites—for example, carboxyl groups on aspartate or glutamate residues or imidazole moieties on histidine residues. The motA proteins, on the other hand, possess a pair of “half-channels,” with one half-channel facing the inside of the cell and the other facing the outside. In Berg’s model, the outside edges of the motA channel protein cannot move past a proton-exchang- ing site on motB when that site has a proton bound, and the center of the channel protein cannot move past an exchange site when that site is empty. As shown in Figure 17.34, these constraints lead to coupling between proton translocation and rotation of the flagellar filament. For example, imagine that a proton has entered the outside channel of motA and is bound to an exchange site on motB (Figure 17.34a). An oscillation by motA, linked elastically to the cell wall, can then position the inside channel over the proton at the exchange site (Figure 17.34b), whereupon the proton can travel through the inside channel and into the cell, while another proton travels up the outside channel to bind to an adjacent exchange site. The restoring force acting on the channel protein then pulls the motA/motB complex to the left as shown (Figure 17.34c), leading to counterclockwise rotation of the disk, rod, and helical filament. The flagellar motor is driven entirely by the proton gradient. Thus, a reversal of the proton gradient (which would occur, for example, if the external medium became alkaline) would drive the flagellar filaments in a clockwise direction. Extending this picture of a single motA/motB complex to the

FIGURE 17.34 ● Howard Berg’s model for coupling between transmembrane proton flow and rotation of the flagellar motor. A proton moves through an outside channel to bind to an exchange site on the M ring. When the channel protein slides one step around the ring, the proton is released and flows through an inside channel and into the cell, while another proton flows into the outside channel to bind to an adjacent exchange site. When the motA channel protein returns to its original position under an elastic restoring force, the associated motB protein moves with it, causing a counterclockwise rotation of the ring, rod, and helical filament.

Dynamics of a tightly coupled mechanism for flagellar rotation. Biophysical Journal 55:905–914)

PROBLEMS

1.The cheetah is generally regarded as nature’s fastest mammal, but another amazing athlete in the animal kingdom (and almost as fast as the cheetah) is the pronghorn antelope, which roams the plains of Wyoming. Whereas the cheetah can maintain its top speed of 70 mph for only a few seconds, the pronghorn antelope can run at 60 mph for about an hour! (It is thought to have evolved to do so in order to elude now-extinct ancestral cheetahs that lived in North America.) What differences would you expect in the muscle structure and anatomy of pronghorn antelopes that could account for their remarkable speed and endurance?

2.An ATP analog, , -methylene-ATP, in which a OCH2O group replaces the oxygen atom between the - and -phosphorus atoms,

is a potent inhibitor of muscle contraction. At which step in the contraction cycle would you expect , -methylene-ATP to block contraction?

3.ATP stores in muscle are augmented or supplemented by stores of phosphocreatine. During periods of contraction, phosphocreatine is hydrolyzed to drive the synthesis of needed ATP in the creatine kinase reaction:

88z phosphocreatine ADP y88 creatine ATP

Muscle cells contain two different isozymes of creatine kinase, one in the mitochondria and one in the sarcoplasm. Explain.

4.Rigor is a muscle condition in which muscle fibers, depleted of ATP and phosphocreatine, develop a state of extreme rigidity and cannot be easily extended. (In death, this state is called rigor mortis, the rigor of death.) From what you have learned about muscle contraction, explain the state of rigor in molecular terms.

5.Skeletal muscle can generate approximately 3 to 4 kg of tension or force per square centimeter of cross-sectional area. This number is roughly the same for all mammals. Because many human muscles have large cross-sectional areas, the force that these muscles can (and must) generate is prodigious. The gluteus maximus (on which you are probably sitting as you read this) can generate a tension of 1200 kg! Estimate the cross-sectional area of all of the muscles in your body and the total force that your skeletal muscles could generate if they all contracted at once.

FURTHER READING

Ahn, A. H., and Kunkel, L. M., 1993. The structural and functional diversity of dystrophin. Nature Genetics 3:283–291.

Allen, B., and Walsh, M., 1994. The biochemical basis of the regulation of smooth-muscle contraction. Trends in Biochemical Sciences 19:362–368.

Amos, L., 1985. Structure of muscle filaments studied by electron microscopy. Annual Review of Biophysics and Biophysical Chemistry 14:291– 313.

Astumian, R. D., and Bier, M., 1996. Mechanochemical coupling of the motion of molecular motors to ATP hydrolysis. Biophysical Journal 70:637– 653.

Berliner, E., Young, E., Anderson, K., et al., 1995. Failure of a single-headed kinesin to track parallel to microtubule protofilaments. Nature 373:718– 721.

Blake, D., Tinsley, J., Davies, K., et al., 1995. Coiled-coil regions in the car- boxy-terminal domains of dystrophin and related proteins: Potentials for protein–protein interactions. Trends in Biochemical Sciences 20:133–135.

Blanchard, A., Ohanian, V., and Critchley, D., 1989. The structure and function of -actinin. Journal of Muscle Research and Cell Motility 10:280–289.

Block, S. M., 1998. Kinesin: What gives? Cell 93:5–8.

Bork, P., and Sudol, M., 1994. The WW domain: A signaling site in dystrophin. Trends in Biochemical Sciences 19:531–533.

Boyer, P. D., 1997. The ATP synthase—A splendid molecular machine.

Annual Review of Biochemistry 66:717–749.

Cooke, R., 1995. The actomyosin engine. The FASEB Journal 9:636–642.

Cooke, R., 1986. The mechanism of muscle contraction. CRC Critical Reviews in Biochemistry 21:53–118.

Coppin, C. M., Finer, J. T., Spudich, J. A., and Vale, R. D., 1996. Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy. Proceedings of the National Academy of Sciences 93:1913–1917.

Davison, M., and Critchley, D., 1988. -Actinin and the DMD protein contain spectrin-like repeats. Cell 52:159–160.

Davison, M., et al., 1989. Structural analysis of homologous repeated domains in -actinin and spectrin. International Journal of Biological Macromolecules 11:81–90.

DeRosier, D. J., 1998. The turn of the screw: The bacterial flagellar motor. Cell 93:17–20.

Eden, D., Luu, B. Q., Zapata, D. J., et al., 1995. Solution structure of two molecular motor domains: Nonclaret disjunctional and kinesin. Biophysical Journal 68:59S–64S.

Engel, A., 1997. A closer look at a molecular motor by atomic force microscopy. Biophysical Journal 72:988.

564 Chapter 17 ● Molecular Motors

Farah, C., and Reinach, F., 1995. The troponin complex and regulation of muscle contraction. The FASEB Journal 9:755–767.

Finer, J. T., Simmons, R. M., and Spudich, J. A., 1994. Single myosin molecule mechanics: Piconewton forces and nanometer steps. Nature 368:113–119.

Fisher, A., Smith, C., Thoden, J., et al., 1995. Structural studies of myosin:nucleotide complexes: A revised model for the molecular basis of muscle contraction. Biophysical Journal 68:19S–26S.

Fisher, A., Smith, C., Thoden, J., et al., 1995. X-ray structures of the myosin motor domain of Dictyostelium discoideum complexed with MgADP BeFx and MgADP A1F4 . Biochemistry 34:8960–8972.

Fleischer, S., and Inui, M., 1989. Biochemistry and biophysics of excita- tion–contraction coupling. Annual Review of Biophysics and Biophysical Chemistry 18:333–364.

Funatsu, T., Harada, Y., Tokunaga, M., et al., 1995. Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution. Nature 374:555–559.

Gilbert, S., Webb, M., Brune, M., and Johnson, K., 1995. Pathway of processive ATP hydrolysis by kinesin. Nature 373:671–676.

Goldman, Y. E., 1998. Wag the tail: Structural dynamics of actomyosin.

Cell 93:1–4.

Gopal, D., Pavlov, D. I., Levitsky, D. I., et al., 1996. Chemomechanical transduction in the actomyosin molecular motor by 2 ,3 -dideoxydidehydro-ATP and characterization of its interaction with myosin subfragment 1 in the presence and absence of actin. Biochemistry 35:10149–10157.

Henningsen, U., and Schliwa, M., 1997. Reversal in the direction of movement of a molecular motor. Nature 389:93–96.

Hirose, K., Lockhart, A., Cross, R., and Amos, L., 1995. Nucleotide-depen- dent angular change in kinesin motor domain bound to tubulin. Nature 376:277–279.

Hoenger, A., Sablin, E., Vale, R., et al., 1995. Three-dimensional structure of a tubulin-motor-protein complex. Nature 376:271–274.

Howard, J., 1996. The movement of kinesin along microtubules. Annual Review of Physiology 58:703–729.

Kabsch, W., and Holmes, K., 1995. The actin fold. The FASEB Journal 9:167–174.

Kabsch, W., et al., 1990. Atomic structure of the actin:DNase I complex.

Nature 347:37–43.

Kikkawa, J., Ishikawa, T., Wakabayashi, T., and Hirokawa, N., 1995. Threedimensional structure of the kinesin head–microtubule complex. Nature 376:274–277.

Kinosita, K., Jr., Yasuda, R., Noji, H., et al., 1998. F1-ATPase: A rotary motor made of a single molecule. Cell 93:21–24.

Koenig, M., and Kunkel, L., 1990. Detailed analysis of the repeat domain of dystrophin reveals four potential hinge segments that may confer flexibility. Journal of Biological Chemistry 265:4560–4566.

Kull, F. J., Sablin, E. P., Lau, R., et al., 1996. Crystal structure of the kinesin motor domain reveals a structural similarity to myosin. Nature 380:550– 555.

Labeit, S., and Kolmerer, B., 1995. Titins: Giant proteins in charge of muscle ultrastructure and elasticity. Science 270:293–296.

Lohman, T. M., Thorn, K., and Vale, R. D., 1998. Staying on track: Common features of DNA helicases and microtubule motors.. Cell 93:9–12.

Löwe, J., and Amos, L., 1998. Crystal structure of the bacterial cell-division protein FtsZ. Nature 391:203–206.

Macnab, R. M., and Parkinson, J. S., 1991. Genetic analysis of the bacterial flagellum. Trends in Genetics 7:196–200.

McLachlan, A., 1984. Structural implications of the myosin amino acid sequence. Annual Review of Biophysics and Bioengineering 13:167–189.

Meister, M., Caplan, S. R., and Berg, H., 1989. Dynamics of a tightly coupled mechanism for flagellar rotation. Biophysical Journal 55:905–914.

Meyhofer, E., and Howard, J., 1995. The force generated by a single kinesin molecule against an elastic load. Proceedings of the National Academy of Sciences

92:574–578.

Molloy, J., Burns, J., Kendrick-Jones, J., et al., 1995. Movement and force produced by a single myosin head. Nature 378:209–213.

Nogales, E., Wolf, S., and Downing, K. H., 1998. Structure of the tubulin dimer by electron crystallography. Nature 391:199–203.

Ohtsuki, I., Maruyama, K., and Ebashi, S., 1986. Regulatory and cytoskeletal proteins of vertebrate skeletal muscle. Advances in Protein Chemistry 38:1–67.

Rayment, I., 1996. Kinesin and myosin: Molecular motors with similar engines. Structure 4:501–504.

Rayment, I., and Holden, H., 1994. The three-dimensional structure of a molecular motor. Trends in Biochemical Sciences 19:129–134.

Saito, A., et al., 1988. Ultrastructure of the calcium release channel of sarcoplasmic reticulum. Journal of Cell Biology 107:211–219.

Smith, C., and Rayment, I., 1995. X-ray structure of the magnesium(II)– pyrophosphate complex of the truncated head in Dictyostelium discoideum myosin to 2.7 Å resolution. Biochemistry 34:8973–8981.

Spudich, J., 1994. How molecular motors work. Nature 372:515–518.

Svoboda, K., Schmidt, C., Schnapp, B., and Block, S., 1993. Direct observation of kinesin stepping by optical trapping interferometry. Nature 365:721–727.

Thomas, D., 1987. Spectroscopic probes of muscle crossbridge rotation.

Annual Review of Physiology 49:691–709.

Trinick, J., 1994. Titin and nebulin: Protein rulers in muscles? Trends in Biochemical Sciences 19:405–409.

Vallee, R., and Shpetner, H., 1990. Motor proteins of cytoplasmic microtubules. Annual Review of Biochemistry 59:909–932.

Wagenknecht, T., et al., 1989. Three-dimensional architecture of the calcium channel/foot structure of sarcoplasmic reticulum. Nature 338:167– 170.

Walker, R., and Sheetz, M., 1993. Cytoplasmic microtubule-associated motors. Annual Review of Biochemistry 62:429–451.

Whittaker, M., Wilson-Kubalek, E., Smith, Jr., et al., 1995. A 35 Å movement of smooth muscle myosin on ADP release. Nature 378:748–753.

Wilson, L., and Jordan, M. A., 1995. Microtubule dynamics: Taking aim at a moving target. Chemistry and Biology 2:569–573.

Worton, R., 1995. Muscular dystrophies: Diseases of the dystrophin–gly- coprotein complex. Science 270:755–756.

The various building blocks are degraded into a common product , the acetyl groups of acetyl-CoA.

Common degradation product

Stage III:

Catabolism converges via the citric acid cycle to three principal end products: water, carbon dioxide, and ammonia.

Glycolysis

Glyceraldehyde-3-phosphate

Pyruvate

Acetyl-

Citric acid cycle

Oxidative phosphorylation

All is flux, nothing stays still.

Nothing endures but change.

HERACLITUS (c. 540–c. 480 B.C.)

OUTLINE

18.1 ● Virtually All Organisms Have the Same

Basic Set of Metabolic Pathways

18.2 ● Metabolism Consists of Catabolism

(Degradative Pathways) and Anabolism

(Biosynthetic Pathways)

18.3 ● Experimental Methods To Reveal

Metabolic Pathways

18.4 ● Nutrition

Special Focus: Vitamins

566

Chapter 18

Metabolism—An Overview

Anise swallowtail butterfly (Papilio zelicans) with its pupal case.

Metamorphosis of butterflies is a dramatic example of metabolic change.

(© 1986 Peter Bryant/Biological Photo Service)

The word metabolism derives from the Greek word for “change.” Metabolism represents the sum of the chemical changes that convert nutrients, the “raw materials” necessary to nourish living organisms, into energy and the chemically complex finished products of cells. Metabolism consists of literally hundreds of enzymatic reactions organized into discrete pathways. These pathways proceed in a stepwise fashion, transforming substrates into end products through many specific chemical intermediates. Metabolism is sometimes referred to as intermediary metabolism to reflect this aspect of the process. Metabolic maps (Figure 18.1) portray virtually all of the principal reactions of the intermediary metabolism of carbohydrates, lipids, amino acids, nucleotides,

and their derivatives. These maps are very complex at first glance and seem to be virtually impossible to learn easily. Despite their appearance, these maps become easy to follow once the major metabolic routes are known and their functions are understood. The underlying order of metabolism and the important interrelationships between the various pathways then appear as simple patterns against the seemingly complicated background.

The Metabolic Map as a Set of Dots and Lines

One interesting transformation of the intermediary metabolism map is to represent each intermediate as a black dot and each enzyme as a line (Figure 18.2). Then, the more than 1000 different enzymes and substrates are represented by just two symbols. This chart has about 520 dots (intermediates). Table 18.1 lists the numbers of dots that have one or two or more lines (enzymes) associated with them. Thus, this table classifies intermediates by the number of enzymes that act upon them. A dot connected to just a single line must be either a nutrient, a storage form, an end product, or an excretory product of metabolism. Also, because many pathways tend to proceed in only one direction (that is, they are essentially irreversible under physiological conditions), a dot connected to just two lines is probably an intermediate in only one pathway and has only one fate in metabolism. If three lines are connected to a dot, that intermediate has at least two possible metabolic fates; four lines, three fates; and so on. Note that about 80% of the intermediates connect to only one or two lines and thus have only a limited purpose in the cell. However, many intermediates are subject to a variety of fates. In such instances, the pathway followed is an important regulatory choice. Indeed, whether any substrate is routed down a particular metabolic pathway is the consequence of a regulatory decision made in response to the cell’s (or organism’s) momentary requirements for energy or nutrition. The regulation of metabolism is an interesting and important subject to which we will return often.

18.1 ● Virtually All Organisms Have the Same

Basic Set of Metabolic Pathways

Table 18.1

Number of Dots (Intermediates) in the Metabolic Map of Figure 18.2, and the Number of Lines Associated with Them

Lines Dots

371

420

511