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

delivery system eminently suited to the needs of the organism. BPG serves this vital function in humans, most primates, and a number of other mammals. However, the hemoglobins of cattle, sheep, goats, deer, and other animals have an intrinsically lower affinity for O2, and these Hbs are relatively unaffected by BPG. In fish, whose erythrocytes contain mitochondria, the regulatory role of BPG is filled by ATP or GTP. In reptiles and birds, a different organophosphate serves, namely inositol pentaphosphate (IPP) or inositol hexaphosphate (IHP) (Figure 15.38).

Fetal Hemoglobin Has a Higher Affinity for O2

Because It Has a Lower Affinity for BPG

The fetus depends on its mother for an adequate supply of oxygen, but its circulatory system is entirely independent. Gas exchange takes place across the placenta. Ideally then, fetal Hb should be able to absorb O2 better than maternal Hb so that an effective transfer of oxygen can occur. Fetal Hb differs from adult Hb in that the -chains are replaced by very similar, but not identical, 146-residue subunits called -chains (gamma chains). Fetal Hb is thus 2 2. Recall that BPG functions through its interaction with the -chains. BPG binds less effectively with the -chains of fetal Hb (also called Hb F). (Fetal -chains have Ser instead of His at position 143, and thus lack two of the positive charges in the central BPG-binding cavity.) Figure 15.39 compares the relative affinities of adult Hb (also known as Hb A) and Hb F for O2 under similar conditions of pH and [BPG]. Note that Hb F binds O2 at pO2 values where most of the oxygen has dissociated from Hb A. Much of the difference can be attributed to the diminished capacity of Hb F to bind BPG (compare Figures 15.35 and 15.39); Hb F thus has an intrinsically greater affinity for O2, and oxygen transfer from mother to fetus is ensured.

Sickle-Cell Anemia

In 1904, a Chicago physician treated a 20-year-old black college student complaining of headache, weakness, and dizziness. The blood of this patient revealed serious anemia—only half the normal number of red cells were pre-

FIGURE 15.38 ● The structures of inositol pentaphosphate and inositol hexaphosphate, the functional analogs of BPG in birds and reptiles.

saturation

80

60

Hb A

Percent O2

40

20

Hemoglobin and Nitric Oxide

Nitric oxide (NO ) is a simple gaseous molecule whose many remarkable physiological functions are still being discovered. For example, NO is known to act as a neurotransmitter and as a second messenger in signal transduction (see Chapter 34). Further, endothelial relaxing factor (ERF, also known as endothelium-derived relaxing factor, or EDRF), an elusive hormonelike agent that acts to relax the musculature of the walls (endothelium) of blood vessels and lower blood pressure, has been identified as NO . It has long been known that NO is a high-affinity ligand for Hb, binding to its heme-Fe2 atom with an affinity 10,000 times greater than that of O2. An enigma thus arises: Why is NO not instantaneously bound by Hb within human erythrocytes and prevented from exerting its vasodilation properties?

The reason that Hb doesn’t block the action of NO is due to a unique interaction between Cys93 of Hb and NO recently described by Li Jia, Celia and Joseph Bonaventura, and Johnathan Stamler at Duke University. Nitric oxide reacts with the sulfhydryl group of Cys93 , forming an S-nitroso derivative:

O CH2OSON O

This S-nitroso group is in equilibrium with other S-nitroso compounds formed by reaction of NO with small-molecule thiols such as free cysteine or glutathione (an isoglutamylcysteinylglycine tripeptide):

H3 NO CO CH2O CH2O CONOCOCONO CH2OCOO

S-nitrosoglutathione

These small-molecule thiols serve to transfer NO from erythrocytes to endothelial receptors, where it acts to relax vascular tension. NO itself is a reactive free-radical compound whose biological half-life is very short (1–5 sec). S-nitrosoglutathione has a half-life of several hours.

The reactions between Hb and NO are complex. NO forms a ligand with the heme-Fe2 that is quite stable in the absence of O2. However, in the presence of O2, NO is oxidized to NO3 and the heme-Fe2 of Hb is oxidized to Fe3 , forming methemoglobin. Fortunately, the interaction of Hb with NO is controlled by the allosteric transition between R-state Hb (oxyHb) and T-state Hb (deoxyHb). Cys93 is more exposed and reactive in R-state Hb than in T-state Hb, and binding of NO to Cys93 precludes reaction of NO with heme iron. Upon release of O2 from Hb in tissues, Hb shifts conformation from R state to T state, and binding of NO at Cys93 is no longer favored. Consequently, NO is released from Cys93 and transferred to small-molecule thiols for delivery to endothelial receptors, causing capillary vasodilation. This mechanism also explains the puzzling observation that free Hb produced by recombinant DNA methodology for use as a whole blood substitute causes a transient rise of 10 to 12 mm Hg in diastolic blood pressure in experimental clinical trials. (Conventional whole blood transfusion has no such effect.) It is now apparent that the “synthetic” Hb, which has no bound NO , is binding NO in the blood and preventing its vasoregulatory function.

In the course of hemoglobin evolution, the only invariant amino acid residues in globin chains are HisF8 (the obligatory heme ligand) and a Phe residue acting to wedge the heme into its pocket. However, in mammals and birds, Cys93 is also invariant, no doubt due to its vital role in NO delivery.

PROBLEMS

1.List six general ways in which enzyme activity is controlled.

2.Why do you suppose proteolytic enzymes are often synthesized as inactive zymogens?

3.First draw both Lineweaver–Burk plots and Hanes–Woolf plots for the following: a Monod–Wyman–Changeux allosteric K enzyme system, showing separate curves for the kinetic response in (1) the absence of any effectors; (2) the presence of allosteric activator A; and (3) the presence of allosteric inhibitor I. Then draw a similar set of curves for a Monod–Wyman–Changeux allosteric V enzyme system.

4.In the Monod–Wyman–Changeux model for allosteric regulation, what values of L and relative affinities of R and T for A will lead activator A to exhibit positive homotropic effects? (That is, under what conditions will the binding of A enhance further A- binding, in the same manner that S-binding shows positive coop-

erativity?) Similarly, what values of L and relative affinities of R and T for I will lead inhibitor I to exhibit positive homotropic effects? (That is, under what conditions will the binding of I promote further I-binding?)

(the fractional saturation of hemoglobin with O2), given P50 and n (see Box on page 484). Let P50 26 torr and n 2.8. Calculate Y in the lungs where pO2 100 torr, and Y in the capillaries where pO2 40 torr. What is the efficiency of O2 delivery under these conditions (expressed as Ylungs Ycapillaries)? Repeat the calculations, but for n 1. Compare the values for Ylungs

Ycapillaries for n 2.8 versus Ylungs Ycapillaries for n 1 to deter-

mine the effect of cooperative O2 binding on oxygen delivery by hemoglobin.

494 Chapter 15 ● Enzyme Specificity and Regulation

6.The cAMP formed by adenylyl cyclase (Figure 15.20) does not persist because 5 -phosphodiesterase activity prevalent in cells hydrolyzes cAMP to give 5 -AMP. Caffeine inhibits 5 -phosphodi- esterase activity. Describe the effects on glycogen phosphorylase activity that arise as a consequence of drinking lots of caffeinated coffee.

7.If no precautions are taken, blood that has been stored for some time becomes depleted in 2,3-BPG. What happens if such blood is used in a transfusion?

FURTHER READING

Creighton, T. E., 1984. Proteins: Structure and Molecular Properties. New York: W. H. Freeman and Co. An advanced textbook on the structure and function of proteins.

Dickerson, R. E., and Geis, I., 1983. Hemoglobin: Structure, Function, Evolution and Pathology. Menlo Park, CA: Benjamin/Cummings.

Gill, S. J., et al., 1988. New twists on an old story: Hemoglobin. Trends in Biochemical Sciences 13:465–467.

Johnson, L. N., and Barford, D., 1993. The effects of phosphorylation on the structure and function of proteins. Annual Review of Biophysics and Biomolecular Structure 22:199–232. A review of protein phosphorylation and its role in regulation of enzymatic activity, with particular emphasis on glycogen phosphorylase.

Johnson, L. N., and Barford, D., 1994. Electrostatic effects in the control of glycogen phosphorylase by phosphorylation. Protein Science 3:1726– 1730. Discussion of the phosphate group’s ability to deliver two negative charges to a protein, a property that no amino acid side chain can provide.

Koshland, D. E., Jr., Nemethy, G., and Filmer, D., 1966. Comparison of experimental binding data and theoretical models in proteins containing subunits. Biochemistry 5:365–385. The KNF model.

8. Enzymes have evolved such that their Km values (or K0.5 values) for substrate(s) are roughly equal to the in vivo concentration(s) of the substrate(s). Assume that glycogen phosphorylase is assayed at [Pi] K0.5 in the absence and presence of AMP or ATP. Estimate from Figure 15.15 the relative glycogen phosphorylase activity when (a) neither AMP or ATP is present, (b) AMP is present, and (c) ATP is present.

Lin, K., et al., 1996. Comparison of the activation triggers in yeast and muscle glycogen phosphorylase. Science 273:1539–1541. Despite structural and regulatory differences between yeast and muscle glyogen phosphorylases, both are activated through changes in their intersubunit interface.

Lin, K., et al., 1997. Distinct phosphorylation signals converge at the catalytic center in glycogen phosphorylases. Structure 5:1511–1523.

Monod, J., Wyman, J., and Changeux, J.-P., 1965. On the nature of allosteric transitions: A plausible model. Journal of Molecular Biology 12:88–118. The classic paper that provided the first theoretical analysis of allosteric regulation.

Rath, V. L., et al., 1996. The evolution of an allosteric site in phosphorylase. Structure 4:463–473.

Schachman, H. K., 1990. Can a simple model account for the allosteric transition of aspartate transcarbamoylase? Journal of Biological Chemistry 263:18583–18586. Tests of the postulates of the allosteric models through experiments on aspartate transcarbamoylase.

Weiss, J. N., 1997. The Hill equation revisited: Uses and abuses. The FASEB Journal 11:835–841.

Appendix to Chapter 15

The Oxygen-Binding Curves of

Myoglobin and Hemoglobin

Myoglobin

The reversible binding of oxygen to myoglobin,

MbO2 34 Mb O2

can be characterized by the equilibrium dissociation constant, K.

If Y is defined as the fractional saturation of myoglobin with O2, that is, the fraction of myoglobin molecules having an oxygen molecule bound, then

The value of Y ranges from 0 (no myoglobin molecules carry an O2) to 1.0 (all myoglobin molecules have an O2 molecule bound). Substituting from Equation (A15.1), ([Mb][O2])/K for [MbO2] gives

and, if the concentration of O2 is expressed in terms of the partial pressure (in torr) of oxygen gas in equilibrium with the solution of interest, then

pO2 K

(In this form, K has the units of torr.) The relationship defined by Equation (A15.4) plots as a hyperbola. That is, the MbO2 saturation curve resembles an enzyme:substrate saturation curve. For myoglobin, a partial pressure of 1 torr for pO2 is sufficient for half-saturation (Figure A15.1). We can define P50 as the partial pressure of O2 at which 50% of the myoglobin molecules have a molecule of O2 bound (that is, Y 0.5), then

(Note from Equation (A15.1) that when [MbO2] [Mb], K [O2], which is the same as saying when Y 0.5, K P50.) The general equation for O2 binding to Mb becomes

pO2 P50

The ratio of the fractional saturation of myoglobin, Y, to free myoglobin, 1 Y, depends on pO2 and K according to the equation

Y

● Oxygen saturation curve fo myoglobin in the form of Y versus pO2 showin P50 is at a pO2 of 1 torr (1 mm Hg).

495

FIGURE A15.2 ● Hill plot for the binding of O2 to myoglobin. The slope of the line is the Hill coefficient. For Mb, the Hill coefficient is 1.0. At log(Y/(1 Y )) 0, log pO2

log P50.

A graph of log (Y/(1 Y )) versus log pO2 is known as a Hill plot (in honor of Archibald Hill, a pioneer in the study of O2 binding by hemoglobin). A Hill plot for myoglobin (Figure A15.2) gives a straight line. At half-satura- tion, defined as Y 0.5, Y/(1 Y ) 1, and log (Y/(1 Y )) 0. At this value of log (Y/(1 Y )), the value for pO2 K P50. The slope of the Hill plot at the point where log (Y/(1 Y )) 0, the midpoint of binding, is known as the Hill coefficient. The Hill coefficient for myoglobin is 1.0. A Hill coefficient of 1.0 means that O2 molecules bind independently of one another to myoglobin, a conclusion entirely logical because each Mb molecule can bind only one O2.

Hemoglobin

New properties emerge when four heme-containing polypeptides come together to form a tetramer. The O2-binding curve of hemoglobin is sigmoid rather than hyperbolic (see Figure 15.21), and Equation (A15.4) does not describe such curves. Of course, each hemoglobin molecule has four hemes and can bind up to four oxygen molecules. Suppose for the moment the O2 binding to hemoglobin is an “all-or-none” phenomenon, where Hb exists either free of O2 or with four O2 molecules bound. This supposition represents the extreme case for cooperative binding of a ligand by a protein with multiple binding sites. In effect, it says that if one ligand binds to the protein molecule, then all other sites are immediately occupied by ligand. Or, to say it another way for the case in hand, suppose that four O2 molecules bind to Hb simultaneously:

Hb 4 O2 34 Hb(O2)4

Then the dissociation constant, K, would be

The experimentally observed oxygen-binding curve for Hb does not fit the graph given in Figure A15.3 exactly. If we generalize Equation (A15.10) by replacing the exponent 4 by n, we can write the equation as

This equation states that the ratio of oxygenated heme groups (Y ) to O2-free heme (1 Y ) is equal to the nth power of the pO2 divided by the apparent dissociation constant, K.

Archibald Hill demonstrated in 1913, well before any knowledge about the molecular organization of Hb existed, that the O2-binding behavior of Hb could be described by Equation (A15.12). If a value of 2.8 is taken for n, Equation (A15.12) fits the experimentally observed O2-binding curve for Hb very well (Figure A15.4). If the binding of O2 to Hb were an all-or-none phenomenon,

1.0

n=4.0

n=2.8

n=1.0

Y 0.5

● A comparison of the experimentally observed O2 curve for Hb yielding a value for n of 2.8, the hypothetical curve if n 4, and the curve if n 1 (noninteracting O2- binding sites).

Mb

Hb

log pO2

● Hill plot (log(Y/(1 Y )) versus log pO2) for Mb and Hb, showing that at log(Y/(1 Y )) 0, that is, Y (1 Y ), the slope for Mb is 1.0 and for Hb is 2.8. The plot for Hb only approximates a straight line.

n would equal 4, as discussed above. If the O2-binding sites on Hb were completely noninteracting, that is, if the binding of one O2 to Hb had no influence on the binding of additional O2 molecules to the same Hb, n would equal 1. Figure A15.4 compares these extremes. Obviously, the real situation falls between the extremes of n 1 or 4. The qualitative answer is that O2 binding by Hb is highly cooperative, and the binding of the first O2 markedly enhances the binding of subsequent O2 molecules. However, this binding is not quite an all-or-none phenomenon.

If we take the logarithm of both sides of Equation (A15.12):

this expression is, of course, the generalized form of Equation (A15.8), the Hill equation, and a plot of log(Y/(1 Y )) versus (log pO2) approximates a straight line in the region around log(Y/(1 Y )) 0. Figure A15.5 represents a Hill plot comparing hemoglobin and myoglobin.

Because the binding of oxygen to hemoglobin is cooperative, the Hill plot is actually sigmoid (Figure A15.6). Cooperativity is a manifestation of the fact that the dissociation constant for the first O2, K1, is very different from the dissociation constant for the last O2 bound, K4. The tangent to the lower asymptote of the Hill plot, when extrapolated to the log(Y/(1 Y )) 0 axis, gives the dissociation constant, K1, for the binding of the first O2 by Hb. Note that the value of K1 is quite large ( 102 torr), indicating a low affinity of Hb for this first O2 (or conversely, a ready dissociation of the Hb (O2)1 complex). By a similar process, the tangent to the upper asymptote gives K4, the dissociation constant for the last O2 to bind. K4 has a value of less than 1 torr. The K1/K4 ratio exceeds 100, meaning the affinity of Hb for binding the fourth O2 is over 100 times greater than for binding the first oxygen.

The value P50 has been defined above for myoglobin as the pO2 that gives 50% saturation of the oxygen-binding protein with oxygen. Noting that at 50% saturation, Y (1 Y ), then we have from Equation (A15.13).

● Hill plot of Hb showing its nonlinear nature and the fact that its asymptotes can be extrapolated to yield the dissociation constants, K1 and K 4, for the first and fourth oxygens.

No single thing abides but all things flow. Fragment to fragment clings and thus they

grow

Until we know them by name.

Then by degrees they change and are no more the things we know.

LUCRETIUS (ca. 94 B.C.–50 B.C.)

500

Chapter 16

Mechanisms of

Enzyme Action

Like the workings of an ancient clock, the details of enzyme mechanisms are at once complex and simple. (David

Parker/Science Photo Library/Photo Researchers, Inc.)

Although the catalytic properties of enzymes may seem almost magical, it is simply chemistry—the breaking and making of bonds—that gives enzymes their prowess. This chapter will explore the unique features of this chemistry. The mechanisms of hundreds of enzymes have been studied in at least some detail. In this chapter, it will be possible to examine only a few of these. Nonetheless, the chemical principles that influence the mechanisms of these few enzymes are universal, and many other cases are understandable in light of the knowledge gained from these examples.