5 Hemoglobin Disorders

Session Level Objectives (SLOs): after completing the session, students will be able to:

SLO 1. Explain the diversity of protein–protein and protein–ligand interactions.

SLO 2. Understand how the quaternary structure of hemoglobin and explain the function of the heme prosthetic group.

SLO 3. For a general ligand-receptor pair, be able to explain and calculate the relationship between kon, koff, and KD.

SLO 4. Describe the mechanistic bases of hemoglobinopathies.

SLO 5. Explain the key properties of enzymes. Explain why many enzymes contain bound prosthetic groups.

SLO 6. Explain and calculate the relationships between kcat, Km, and Vmax. Michaelis-Menten enzyme kinetics

SLO 1. Explain the diversity of protein–protein and protein–ligand interactions.

In this session, we begin to think seriously about how proteins do their jobs. Proteins come in a dazzling variety of shapes, sizes, abundances, and tissue distributions.

Nearly without exception, all proteins are functionally similar: what proteins do is recognize specific chemical entities, and bind — stick — to them.

That’s it. That’s almost (though not quite) the whole deal. Proteins stick to specific things. Antibodies stick to antigens to mediate immune responses during infection. Cell adhesion molecules allow cells to stick to each other and to extracellular matrix proteins. Extracellular matrix proteins stick to each other, and to cells. Transcription factors recognize and stick to specific enhancer sequences in our genes. Odorant receptors stick to specific volatile molecules (from perfume to putrescine). Hormone receptors stick to insulin, estrogen, or other hormones.

Enzymes are also most simply understood through their ability to stick to things. They stick to their substrates, and they stick to transition states more tightly, favoring the formation of those otherwise unfavored states, and accelerating reaction rates. Enzymes often bind products less tightly, allowing their dissociation (release) from the enzyme.

Within membranes, ion channels, transporters, and pumps are again sticking to substrates and using a series of binding steps to move things from one side of a membrane to the other.

Molecular motors like myosin do the same thing again. Myosin sticks and un-sticks to the actin thin filament. The order of myosin-actin sticking-unsticking is coupled to myosin sticking (binding) to ATP, to the ATP hydrolysis transition state, and finally to the release of ADP and Pi. Here, coupling of two binding cycles allows energy derived from ATP hydrolysis to make myosin’s binding and un-binding to actin directional — and that is the power stroke that makes our muscles contract.

With a general quantitative description of a protein’s binding characteristics we can understand an enormous amount about what a protein does, how it does it, and how it can fail in its functions, leading to pathology. The same concepts, as we will see in the next session, allow us to think about how drugs interact with their molecular targets.

After all, most drugs are just chemical entities that particular proteins recognize, and stick to.

SLO 2. Understand the structure of hemoglobin and explain the function of heme in hemoglobin.

We begin this exploration with a protein we’ve already seen. Hemoglobin (Hb) is a protein present at

enormously high concentration in the cytoplasm of red blood cells. The most important (but not only) thing hemoglobin sticks to is molecular oxygen (O2).

Structure of Hb

1. Adult Hb (HbA) is a complex of four polypeptides: two α chains and two β chains (α2β2).
2. Fetuses and infants have fetal HbF containing two γ (gamma) chains instead of β chains (α2γ2). The α, β, and γ chains are not identical, but they have very similar primary sequences and a nearly identical, all α-helical, tertiary fold.
3. Terminology note: α -helices and β-sheets are not the same as the α and β chains of Hb.
4. Separate genes encode mRNA templates for the various Hb chains.
5. Each of the four chains cradles one heme molecule (Figs 1 and 2): a porphyrin ring that coordinates an ion of iron (Fe2+) at its cent
   

   

   er. The bound heme is an example of a prosthetic group — a non- amino acid bound to an enzyme and required for its activity. As we’ll soon see, vitamins often serve as prosthetic groups in enzymes.

6. Within each subunit (or chain), amino acid side-chains and the bound heme operate together to bind one O2 molecule (Fig. 2). Thus, the binding capacity of a hemoglobin heterotetramer is 4 O2.
7. It follows that a one Hb tetramer can be ¼, ½, ¾, or completely saturated with O2.
8. With huge numbers of Hb molecules, O2 saturation of the population can be anywhere from 0 to nearly 100%.

Function: what Hb needs to do

As erythrocytes pass through our lungs, their Hb binds O2. The erythrocytes flow with the blood to our peripheral tissues where they dump the O2. The tricky bit is that Hb needs to hold on to its precious cargo of O2 until it is in a part of the body that’s most in need of O2.

In other words, Hb needs to bias its O2 binding characteristics to accelerate dissociation in relatively hypoxic environments, rather than dumping O2 randomly. This, along with control of vasoconstriction, allows us to maintain a relatively shallow pO2 gradient from our lungs to our fingers, toes, and brain in the periphery.

SLO 3. For a general ligand–receptor pair, be able to explain and calculate the relationship between kon, koff, and KD.

O2 is a ligand, and we can say that HbA is a receptor that binds it. As the partial pressure of oxygen, pO2, increases, the fractional saturation of HbA increases (Fig. 3).

The first thing to notice is this is an ensemble measurement of many molecules of HbA. We note that there is a concentration of O2 where 50% the receptor (HbA) is 50% saturated by its ligand (O2).

1. This concentration is the dissociation constant (K_D), and it indicates how tightly a ligand binds its receptor. For this reason, K_D is also called the affinity constant.
2. K_D has units of concentration. For a gas, we may use partial pressure (units: mm Hg, atmospheres, Pa, pounds per inch² (p.s.i.), etc.). For solutes in liquid we will more often use concentration per volume (M, g/L, etc.).
3. We say that an interaction with a small K_D (say, 10^-9 M) is high–affinity. We say that an interaction with a large K_D (say, 10^-3 M) is low–affinity.
   

   

4. For a simple ligand-receptor pair, L + R ⇌ LR,         KD = ([L][R])/[LR] where [L], [R], and [LR] are the concentrations of the ligand, the receptor, and the complex, at equilibrium (when the concentrations of free and receptor-bound ligand are not changing).
5. KD is an equilibrium constant, which reflects two rates: the rate at which a particular ligand sticks to the receptor (association), and the rate at which that ligand falls off (dissociation). For the simple case, L + R ⇌ LR,      K_D = k_off/k_on       where k_on is the rate constant for association and koff is the rate constant for dissociation. k_on has units of inverse concentration and time (M^-1 s^-1), and K_off has units of inverse time (s-1).
6. Take the time to satisfy yourself that if the association rate constant increases, the receptor-ligand affinity increases. Conversely, if dissociation rate constant increases, the affinity decreases.

Now take another look at the shape of the saturation curve for HbA. What does the shape of the curve tell us? We will be thinking through this question in class.

SLO 4. Describe the mechanistic bases of hemoglobinopathies.

Diseases caused by mutations that alter hemoglobin function are probably the most prevalent and best-understood of all Mendelian genetic disorders. At least 1 in 15 people carry genetic variants that contribute to hemoglobin-related disorders. To understand the hemoglobinopathies we need to start by looking at the genes that encode the various Hb chains, and their expression patterns (Fig. 4).

1. First, note that during gestation, Hb is initially produced in the yolk sac and then in the liver. Toward the end of gestation, Hb production gradually switches to the bone marrow.
2. Concomitant with the changes in the locations of Hb production, the chain types produced are also changing. The major form of fetal and infant Hb, HbF (α2γ2), is gradually supplanted by adult Hb A (α2β2).
   

   

    

3. These changes are called Hb switching. They represent a classical example of developmental regulation of gene transcription. The mechanisms through Hb switching occurs include both the types of mechanisms we have already seen, and “epigenetic” changes in DNA packaging (chromatin regulation) that we’ll see in more detail later.
4. When we look at the arrangement of the genes that encode Hb chains, we see that they are arranged in clusters: the β-like loci sit together on chromosome 11, and the α-like loci sit together on chromosome 16.
5. Remarkably — and unusually — each set of genes is arranged in its temporal order of expression.

Sickle cell disease (hemoglobin S disease)

Sickle Cell Anemia is the most prevalent single hemoglobinopathy. It falls within the broader class of hemolytic anemias.

Note — Cyanosis vs. Pallor. You cannot manifest red (or blue) color without blood. Pallor (being pale) reflects anemia. Cyanosis (being blue) indicates that blood is poorly oxygenated. As mentioned above, HbS carries O2 normally, but leads to clogging of capillaries and hemolysis, the latter resulting in anemia.

1. Sickle disease results from a single nucleotide missense substitution, βS, that changes a codon for glutamate to a codon for valine at amino acid position 6 in the Hb β–chain (β–globin).
   

   

2. The β S mutation has no effect on the ability of Hb to carry O2.
3. Heterozygote carriers of the sickle allele, referred to as βS, generally present few or no symptoms. Extreme physical exertion can lead to rhabdomyolisis.
4. Heterozygote carriers exhibit partial resistance to malaria. The βS allele appears to have appeared de novo multiple times. Its prevalence is elevated in populations in the Mediterranean, Africa, and southern Asia, all areas with endemic malaria.
5. Heterozygous carriers of the sickle trait produce a mix of Hb tetramers: α2A β2A (normal HbA), α2A β2S (sickle HbS), and α2A βA βS.
6. Homozygotes with sickle cell disease produce mainly α2A β2S (sickle) Hb tetramers.
7. The O2-free form of HbS, deoxyHbS (deoxy- HbS), is five times less soluble than deoxyHbA.
8. At the high concentrations of HbS that are present in homozygotes, deoxy–HbS tetramers assemble into long, higher-order filaments. These deoxy-HbS filaments distort the normal rounded shape of erythrocytes, leading to clogging of capillaries. The fibers can also puncture the cell’s membrane, causing erythrocyte lysis (hemolysis).
9. Sickle crisis is an episode of extreme pain lasting hours to days. Generally, crisis is thought to result from sickled erythrocytes blocking blood flow, particularly to bones. A short (2 min.) video on sickle crisis is here:

https://www.nejm.org/do/10.1056/NEJMdo005311/full/

This is one of many examples you’ll see of protein folding diseases.

Other hemoglobinopathies

In addition to S disease there are a large number of mutations in the various Hb chains that can lead to disease. These fall into three categories:

1. Structural hemoglobinopathies. These disorders are generally caused by missense mutations that alter the primary structure of Hb chains. Sickle (HbS) disease is an example.
2. Thalassemias. These disroders are caused by imbalances in the amounts of α- and β-chain synthesis, degradation, or Hb tetramer assembly, leading to excess production of unassembled globin chains. Note: some structural hemoglobinopathies can result in thalassemia.
3. Hereditary persistence of fetal hemoglobin (HSPS). These are regulatory disorders in which the switch from HbF to HbA fails to occur in early childhood. HSPS by itself does not lead to major pathology, but it can be a strong genetic modifier of other hemoglobinopathies and thalassemias.

An absolutely key point is that there are two identical copies of the Hb α-chain gene on chromosome 16 (See fig. 4). This means that most people have four copies of the α-chain gene. Consequently, recessive disorders of Hb β–chains are far more frequent, and tend to be more severe, than disorders of the Hb α-chains.

Moreover, because of the temporal order of Hb chain expression (Fig. 3), disorders of Hb β–chains tend to manifest only in childhood (recall that HbA is α2β2), while disorders of Hb α-chains can begin manifesting prenatally (HbF is α2γ2).

Structural hemoglobinopathies

For the Hb β–chain (β-globin) alone, mutations leading to the synthesis of well over 700 different structural variants of the protein have been identified. Additional variants in other globins can also cause disease.

The Hb structural variants were originally named with letters (HbS, HbE, etc., and later, they were named by the location where the carriers of the mutations were identified (Hb Hammersmith, etc.). Structural mutations can lead to changes in the O2 saturation curve (and cyanosis, as in Hb Hammersmith), changes in Hb solubility, and hemolysis (as in HbS), to thalassemias, or combinations of these defects.

In addition, the iron in the heme center can be oxidized by bound O2, from ferrous (Fe2+) to ferric (Fe3+) iron. Hb containing an oxidized heme center cannot bind O2, and is called methemoglobin. An enzyme, methemoglobin reductase converts the heme iron to the ferrous (Fe2+) state, and restores its ability to carry O2.

Mutations that impair methemoglobin reductase cause gradual conversion of Hb to methemoglobin, resulting in cyanosis. Moreover, some structural variants of Hb (such as Hb Hyde Park) cannot productively engage with methemoglobin reductase, and these also gradually convert into methemoglobin (also leading to cyanosis).

Thalasseimias

A vast variety and number of mechanisms and mutations cause imbalances in globin chain abundance. The resulting diseases occur through a variety of mechanisms, with diverse presentations and severities. Collectively, these diseases are called thalassemias.

1. α–thalassemia. Recall that there are two α-globin genes on chromosome 16 (Fig. 4). α-thalassemias most commonly arise through deletion of entire α1 or α2 genes.
   • The silent carrier genotype is –α/αα, resulting in 75% of normal α-globin production.
   • The α-thalassemia trait occurs in two forms: – –/αα, and –α/–α. These genotypes result in 50% of normal α-globin production.
   • Simple α-thalassemia disease is associated with the – –/–α genotype. Only 25% of the normal α-globin amount is produced, and an excess of Hb β4 tetramers are produced. This variant is also called HbH or Hb Bart’s.
   • The – –/– – genotype results in assembly of Hb γ4 tetramers. Because this genotype is embryonic lethal, the switch from γ to β expression never occurs.
2. β–thalassemia. A wide variety of mutations can cause reduced production of β globin chains, and β-thalassemia. We review these because they illuminate and summarize many of the ways that mutations can change protein production.
   • The locus control region (LCR; Fig. 4) is needed for transcription of the entire set of β-like globin chains. Deletions within the LCR can decrease or eliminate transcription of all of the β-like genes.
   • Similarly, mutations in the β-chain promoter can impair transcription of the β-chain mRNA alone, but leave transcription of the other β-chain genes intact.
   • Mutations in the β-chain transcription unit can prevent formation of a functional mRNA template:
     • Defects in 5´cap addition;
     • mRNA splicing defects;
     • Failure of poly–A tail addition;
     • Point mutations that eliminate the start codon;
     • Insertions or deletions (indels) that cause frameshifts;
     • Introduction of premature stop codons (nonsense mutations).
     • Missense mutations (like those in the structural hemoglobinopathies) can also cause instability β-chain protein and its destruction by the protein quality control system.
3. As with sickle cell trait (HbS), heterozygotes carrying the α- and β-thalassemia traits are partially protected from malaria. These traits (and hence, the associated diseases) occur at elevated levels in populations affected by endemic malaria. Because most disease alleles are in carriers compared to affecteds for recessive disorders (2pq>>q2), a fitness advantage to carriers often outweighs the loss of fitness among those with disease. (As used here, fitness refers to survival to reproduction.)

SLO 5. Explain the key properties of enzymes. Explain why many enzymes contain bound prosthetic groups. Describe enzymatic co-factors and how deficiency of these factors leads to disease.

Enzymes

Enzymes are highly selective catalysts.

1. Enzymes bring together specific substrate molecules in specific geometries, and accelerate chemical reactions that convert the substrates into specific products.
2. As catalysts, enzymes cannot change the thermodynamics — the overall free energy balance — of a reaction
   

   

   (Fig. 6).

3. Instead, enzymes change the kinetics of the reaction. They do this by reducing activation energy barriers that would otherwise prevent the reaction from occurring at a biologically relevant rate.
4. By bringing specific substrates together in the appropriate geometry, and by shielding intermediates from reactive non-substrate molecules, enzymes suppress the formation of off– pathway products.
5. The vast majority of enzymes are proteins. (But, as we saw with the ribosome, a small critical subset of enzymes have catalytic centers made out of RNA.)
6. Many enzymes use covalently or non-covalently bound prosthetic groups to control the electronic environment within their active sites, promoting catalysis. Prosthetic groups are sometimes, but not always, vitamins. Thus, vitamin deficiency often leads to compromised function in specific enzymes, leading to metabolic or structural pathologies.
7. In summary, we can think of enzymes as receptors that bind ligands (substrates) in highly specific ways, to promote specific chemical reactions among those substrates.

SLO 6. Explain and calculate the relationships between kcat, Km, and Vmax. Michaelis-Menten enzyme kinetics

As with ligand-receptor interactions, a small number of parameters provides a vivid description of an enzyme’s critical properties.

1. V is the reaction rate at which substrate is converted to product, under some specified set of conditions (substrate concentration, temperature, pH, etc.).
2. The V_max is the maximum rate at which the enzyme can convert substrate to product. V_max occurs when the substrate is at a sufficiently high concentration that its availability is not rate-limiting.
3. The K_M, or Michaelis–Menten constant, is the concentration of substrate at which the rate of product formation per molecule of enzyme is half-maximal. Thus, at a substrate concentration [S], where V = V_max/2,   K_M = [S]
4. K_M is closely analogous to K_D, because each refers to the concentration of ligand or substrate where 50% of the receptor or enzyme is occupied.
5. k_cat is the rate constant for the enzyme-catalyzed conversion of the enzyme-bound substrate to product: For the scheme in Fig. 3, k_cat is the rate for ES ⟶ E+P. kcat is also called the enzyme’s turnover rate.
6. The catalytic specificity of an enzyme for any given substrate can be defined as a ratio, k_cat /K_M
7. By comparing k_cat /K_M ratios for one enzyme and various substrates, we can learn how selective an enzyme is. For this reason k_cat /K_M is often called the specificity constant.
8. For example: the active site pockets of DNA polymerase enzymes have high affinity (small K_M) for dNTP (DNA) nucleotides, but extremely low affinity (very large KM) for NTP (RNA) nucleotides. This is because DNA polymerase active sites are usually shaped so that the 2´-OH group of dNTPs does not fit within the pocket.
9. Thus for DNA polymerases k_cat /K_M for dNTPs is relatively large, while k_cat /K_M for NTPs is very small. We therefore say that DNA polymerases are selective for dNTP substrates versus NTP substrates.
10. As we’ll see in following sessions on Pharmacology, competitive enzyme inhibitors have high affinities for enzyme active sites (low K_M), but small — or zero — k_cat. Competitive inhibitors “clog” an enzyme’s active site, preventing legitimate substrates from binding.