BIOCHEMISTRY - L. Stryer - 1984

VOLUME 1

PART I. CONFORMATION AND DYNAMICS

CHAPTER 5. MOLECULAR DISEASES: SICKLE-CELL ANEMIA

5.9. High Frequency of the Sickle-Cell Gene Due to Its Protective Effect Against Malaria

The frequency of the sickle-Cell Gene reaches 40% in certain areas of Africa. Until recently, individuals homozygous for this gene died before reaching maturity; it follows that the high frequency of this gene in the population could be maintained only under strong natural Selection pressure. James Neel suggested that the heterozygous state must confer some advantages compared to homozygosity for either the normal or the defective gene. Indeed, Anthony Allison discovered that carrying the sickle-cell trait provides protection against the most severe and usually fatal form of malaria. A clear correlation has been revealed between the distribution of malaria and the frequency of the sickle-cell gene in Africa (Fig. 5.15). This phenomenon is a classic case of balanced polymorphism: heterozygotes are resistant to malaria and do not suffer from Sickle-Cell Anemia, whereas individuals homozygous for the normal allele are susceptible to malaria.

Class="center">Fig. 5.15. Distribution of the sickle-cell gene in Africa. The high frequency of this gene is restricted to regions where malaria is a major cause of mortality

5.10. Strategy for Drug Discovery in the Treatment of Sickle-Cell Anemia

Insights into the Molecular Mechanism of sickle-cell anemia now allow us to move toward the discovery of specific, non-toxic drugs that prevent or delay The Development of the clinical manifestations of the disease. The search is being pursued in three directions. One group of promising agents consists of substances that inhibit The formation of Hemoglobin S fibers. Synthetic oligopeptides, whose Amino Acid Sequence corresponds to the N-terminal region of the β^S chain and other contact sites in the hemoglobin S molecule, are being evaluated as stereospecific inhibitors of gelation. A second group of investigated substances comprises compounds that increase the affinity of hemoglobin S for oxygen and thereby lower the concentration of its deoxy form. For example, cyanate penetrates erythrocytes and increases hemoglobin's oxygen affinity through the irreversible carbamoylation of α-amino groups. Hemoglobin reacts rapidly with cyanate because HN=C=O (isocyanic acid) in its unionized form is a reactive analogue of O=C=O. Recall that the terminal amino groups of hemoglobin participate in the Reversible Binding of CO₂ (Section 4.15). Great hopes were pinned on The Use of cyanate in the Treatment of sickle-cell anemia until extensive clinical trials revealed its adverse toxic side effects. For instance, some patients treated with cyanate developed peripheral neuropathy, presumably because carbamoylation affects not only hemoglobin but other Proteins as well. The search for less toxic hemoglobin modifiers is currently underway. A third potential approach to treating sickle-cell anemia is to reduce the overall concentration of hemoglobin S in erythrocytes. This can be achieved, for example, by increasing cell volume. Since The rate of deoxyhemoglobin fiber formation depends heavily on its concentration, even a slight increase in erythrocyte volume would produce a pronounced therapeutic effect. It follows that the ionic pumps and channels of The erythrocyte membrane are potential targets for therapeutic agents used in the treatment of sickle-cell anemia.

5.11. Molecular Pathology of Hemoglobin

Examination of patients with symptoms of sickle-cell anemia, as well as electrophoretic analysis of Hemoglobins from healthy individuals, has revealed more than 100 abnormal hemoglobins. In northern Europe, heterozygosity for the hemoglobin A variant occurs in 1 out of 300 individuals. The frequency of any given mutant allele is usually less than 10^-4; this is several orders of magnitude lower than the frequency of the sickle-cell gene in malaria-endemic areas. In other words, most abnormal hemoglobins confer no evolutionary advantage in natural selection. As a rule, the presence of abnormal hemoglobins either has no effect on human health or proves detrimental.

Several types of abnormal hemoglobins are distinguished.

1. Altered exterior of the molecule. Almost all Amino Acid Substitutions On the surface of the hemoglobin molecule are harmless. Hemoglobin S is a striking exception.

2. Altered active center. In this case, oxygen binding

does not occur in the defective subunit, because structural changes near the heme directly affect oxygen binding.

3. Altered tertiary Structure. Amino acid substitutions prevent the Formation of the normal molecular conformation. Such hemoglobins are typically unstable.

4. Altered quaternary structure. Certain Mutations affecting contact region sites lead to the loss of allosteric properties. As a result, the affinity of such hemoglobins for O₂ is impaired.

5.12. Hemoglobin M: A Product of Active Center Mutation

Replacement of the proximal or distal Histidine in the heme group by Tyrosine leads to the stabilization of the oxidized (ferric) form of heme, which is incapable of binding oxygen (Fig. 5.16). In this case, the ionized side chain of tyrosine forms a complex with the oxidized iron of the heme. This substitution can occur in either the α- or the β-chain. Indeed, all four mutant variants have been discovered. Mutant hemoglobins in which two Hemes in the molecule are permanently in the ferric state are termed hemoglobin M. The letter M indicates that the mutated chains are in the methemoglobin (ferricytochrome/ferrihemoglobin) form. Individuals with hemoglobin M typically exhibit cyanosis. The disorder occurs only in heterozygous individuals, since homozygosity for this trait is almost invariably lethal.

Fig. 5.16. Replacement of the proximal histidine (F8) by tyrosine leads to the formation of hemoglobin M. The negatively charged oxygen atom of tyrosine binds to the iron atom in the ferric state. Water, rather than O₂, occupies the sixth coordination position

5.13. Polar Groups in the Heme Pocket Weaken Its Binding to the Polypeptide Chain

Another type of mutation affecting the active center is the replacement of a nonpolar group by a polar one within the heme pocket. There are 60 interatomic contacts between the heme and the polypeptide chain, and these contacts are nonpolar in nature. Because these nonpolar interactions are stably conserved in normal hemoglobins across various animal species, it is believed that most of them are essential for the functioning of the hemoglobin molecule. Indeed, mutations at Heme-binding sites almost always lead to adverse consequences. Consider Hemoglobin Hammersmith as an example. In this hemoglobin, Serine replaces phenylalanine CD1 (Fig. 5.17). This substitution disrupts heme binding. The explanation for this is likely that the presence of the polar serine residue facilitates the penetration of water into the cavity normally occupied by the heme.

Fig. 5.17. Localization of phenylalanine CD1, one of the invariant residues in hemoglobin. The aromatic ring of this phenylalanine is in contact with the heme