ALAD Inhibition

ALAD Function and Purpose

δ-aminolevulinic acid dehydrogenase (ALAD) (also called Porphobilinogen Synthase (PBGS)) is an enzyme considered to be one of lead’s main targets.³⁷  The primary enzymatic function of ALAD is to catalyze the second reaction in the heme biosynthetic pathway (Figure 1). In this step, two molecules of δ-aminolevulinic acid (ALA) are condensed to produce porphobilinogen (PBG) (Figure 4).³⁸ Heme is an essential molecule for most archaea, bacteria, and eukaryotes. However, accumulation of the pathway’s intermediates, which occurs when a step is inhibited, can be toxic.³⁹ Lead is known to inhibit the activity of ALAD, as noted in workers exposed to lead.³⁷ Lead ions inhibit ALAD by replacing the zinc bound to the ALAD’s triple-cysteine binding site.²⁷ Since ALAD is necessary for the second step of heme biosynthesis, its inhibition will cause a build up of ALA in the cell, which is responsible for many negative effects of lead (see Figure 5 of the ROS Generation and ALA page for a discussion of consequences specifically related to ALA autooxidation). 

ALAD Structure

ALAD is an octamer composed of eight monomers that organizes itself into four dimers.⁴⁰

The tertiary structure of the ALAD monomer is mainly characterized by the TIM-barrel which contains an eight-membered β-sheet surrounded by eight α-helices. The ALAD active site as seen in Figure 3, is located at the C-terminal ends of the β-strands that create the TIM-barrel loop.⁴⁰ The TIM-barrel at the C- terminal end and the extended arm region at the N-terminal end are key components of the ALAD structure that drive the quaternary interactions. The ALAD dimer forms when the arm region of one monomer folds itself over the neighboring monomer’s TIM barrel.⁴⁰ This crosslinking of the monomers to form a dimer is the strongest binding force in the ALAD octamer.⁴² Once the four dimers of the ALAD octamer have formed, they orient their long axes to be parallel, but slightly tilted, to the vertical 4-fold axis as seen in Figure 1.⁴⁰ The dimer-dimers interactions occur principally with the helical segments of the arm regions on the surface of one dimer interacting with the N-terminal end of the neighboring dimer’s β- barrel; this forms an exposed active site on the ALAD octamer surface.⁴³

The ALAD active site has unique features that share commonalities with Schiff base aldolases (class I) and metallo-aldolases (class II).⁴⁴ The ALAD catalyzed Knorr-type condensation reaction shown in Figure 4 is an asymmetric condensation of two molecules of ALA yielding polypyrrole porphobilinogen and two water molecules.⁴⁴ The substrate is classified as “A side” and “P-side” based on whether they contribute the acetyl/amino or the propionyl/pyrrole-nitrogen portion of the porphobilinogen product.⁴⁴ 

This reaction demonstrates class I and class II aldolase features.⁴⁴ In the active site at the C-terminal end of the β- barrel, the 4- keto group from a molecule P-side substrate and Lys 252 form a Schiff base – demonstrating class I aldolase function.⁴⁰ The A-side substrate binding requires bound metal ions that facilitate the intra-substrate C-C bond formation via polarization of the C-4 carbonyl – demonstrating mechanistic qualities of class II aldolases.⁴⁰  Illustrated in Figure 3, Lysine 199 and 252 side chains lie in the cavity formed by the terminal loops of the β- barrel. Lys 252 is more hydrophobic than Lys 199 and forms a Schiff base with the P-side of the substrate molecule.⁴² It is predicted from the polarity of the environment that Lys 199 contains a positive charge which lowers the pKa for the Lys 252 Schiff base.⁴⁰ The ALAD active site also contains tyrosines and polar residues dominated by hydrogen bonding interactions.⁴⁰ 

Zinc Binding Site and Lead Inhibition

Zinc is essential for the function of ALAD. Eight Zn(II) ions are associated with the homo-octamer.²⁷ Zinc binds at two metal binding sites on ALAD, rather than at the enzyme’s active site. Situated inside the loop connecting β5 and α2 at the end of the TIM barrel is the zinc primary binding site, as depicted in Figures 5.⁴⁰ The second metal binding site is adjacent to a disordered loop region that is instrumental for substrate binding by undergoing a conformational change and acting as an active site cover when the binding occurs.⁴⁰ 

The two metal binding sites are called the ZnA site (secondary) and ZnB site (primary); the ions at the sites are called ZnA and ZnB, respectively. ZnB interacts with a cluster of cysteine residues: Cys-122, Cys-124, and Cys-132,²⁷ and zinc is coordinated with approximately tetrahedral geometry.⁴⁰ 

ZnA interacts with His-131 and Cys-223; this site has lower occupancy than ZnB.²⁶ It is not yet fully understood how zinc contributes to catalysis. ZnB is in proximity to an active site lysine, but its coordination environment does not emulate that of catalytic zinc; the environment of ZnA is more similar to that of catalytic zinc, but it is farther from the lysine.²⁷ One possibility is that ZnB coordinates with the 4-keto group of A-side ALA, which would aid in the polarization of its carbonyl functionality (Figure 7).⁴⁰ The binding of the A-side ALA does require bound zinc ions,⁴⁰ and there is a consensus that zinc is important for binding this substrate.⁴⁵

By studying the kinetic parameters of ALAD variants ‘MinusZnA’ and ‘MinusZnB’, Jaffe et al. found that the absence of the ZnB site is linked to greatly diminished catalytic capabilities, while the absence of the ZnA site did not greatly impact activity.²⁷ This suggests that the ZnB site may be more important for catalytic activity. They also found that both variants are sensitive to lead, though the kinetics of inhibition differ between sites.²⁷ At the ZnB (PbB) site, there is lead inhibition during steady-state turnover, whereas the PbAB site (hybrid of ZnA and ZnB sites) is disfavored during turnover.²⁷ 

Lead is able to interact with ALAD’s metal binding sites and displace zinc.²⁷ The binding of a metal to a ligand is based upon the metal’s size and geometric coordination preferences, as well as the type of Lewis acid (“hard” or “soft” classification). Briefly, the Hard and Soft Acid Base Theory states that soft acids and soft bases (characterized by low charge density) are attracted to each other and hard acids are attracted to hard bases (characterized by high charge density).⁴⁶ The thiolate of cysteine is considered a soft base due to its low charge-to-radius ratio, and zinc is an intermediate acid, so it is compatible with a soft base. Also, the metal binding site accommodates a certain size and facilitates tetrahedral geometry.⁴⁰ While these specifications mean that the other metal ions present in an organism will not occupy the binding site, lead, which is absent in biological systems, has the appropriate attributes to do so. At the “PbB” site, lead uses the three cysteines Cys-122, Cys-124, and Cys-132 and its own lone pair of electrons in tetrahedral coordination; similarly, at the “PbAB” site, lead uses Cys-124, Cys-132, and Cys-223 as ligands.²⁷ The PbAB site, the tight-binding inhibitory Pb(II) site, is formed by one of the ZnA ligands and two of the ZnB ligands.²⁷ 

Thus, the presence of lead is inhibitory in two respects. Most plainly, it displaces zinc from the metal binding site, precluding catalytic activity that requires zinc (presumably at the ZnB site). Also, the lone pair of lead completes its tetrahedral coordination geometry, while zinc coordinates with the three cysteines and the substrate as its fourth coordinate bond.³⁰ This discrepancy could disrupt binding interactions between ALAD and the substrate ALA. 

Impacts on the Nervous System

Inhibition of ALAD has many physiological impacts beyond the direct production of reactive oxygen species (see Free Radicals and Oxidative Stress). As aforementioned, ALAD inhibition blocks the heme biosynthesis pathway.²⁹ Without properly functioning heme, erythrocytes cannot bring sufficient oxygen to body tissues, which is particularly troublesome for the brain which requires a large amount of oxygen. 

The buildup of ALA itself is also harmful to the body. ALA can accumulate in nervous tissue, blood, heart, liver, kidney, spleen, gut, and fat tissues.²⁹ In lead-exposed workers, plasma concentrations of ALA were about 10 µM, compared to 0.1 µM in the control group.⁴⁸ This blood accumulation means that the ALA can be brought to anywhere in the body, and the molecule can then enter the central nervous system, as it can cross the blood brain barrier, where it causes impairment of synaptic transmission.²⁹ It accomplishes this impairment via competition for GABA presynaptic receptors.⁴⁸ Usually, GABA receptors reduce Ca²⁺ concentration, but in the presence of ALA, cortical synaptosomes are observed to have increased Ca²⁺ uptake.⁴⁸ This may result from synaptosomal membrane alterations such as lipid and protein oxidation as indicated by partial protection provided by antioxidants as well as increased thiobarbituric acid reactive substances (TBARS) formation and lack of nifedipine effect on Ca²⁺ uptake.⁴⁸ TBARS are formed as a byproduct of lipid peroxidation, and nifedipine is a calcium channel blocker.

This intake of Ca²⁺ impacts the mechanisms within the cell, where Ca²⁺ homeostasis is vital for neuronal functioning and neuronal ATP production for native and mature neurons.⁴⁹ The increased concentration of Ca²⁺ within the cell leads to irreversible cell damage including the uncoupling of mitochondrial oxidative phosphorylation.⁴⁸ Incubation for 15 min with ALA concentrations of 0.5 or 1mM caused a mitochondrial membrane potential dissipation of about 40%, significantly impacting the mitochondrial function.⁴⁸ Intra synaptosomal ALA-generated ROS also damaged the mitochondrial membrane. It is thought that the ROS damage is strengthened by increased Ca²⁺ uptake.⁴⁸ The mitochondrial permeabilization is indicated experimentally by the collapse of the transmembrane electrical potential, Ca²⁺ release, and mitochondrial swelling.⁴⁸ 

Damaged mitochondria leads to decreased ATP concentration and increased concentration of ROS.⁴⁹ The brain is responsible for 20% of total body oxygen consumption, so it is especially susceptible to oxidative stress.⁴⁸ The mitochondria is crucial in regulating cell death by apoptosis or excitotoxicity, which are significant grounds for neuronal cell death.⁴⁹ This has been hypothesized to underlie the central nervous system syndrome experienced by those who have had prolonged exposure to lead, as well as psychomotor agitation, hallucination, seizures, and depressive episodes.⁴⁸

Some drugs have been shown to mitigate the effects of ALA-generated radical damage. Vitamin E provided significant protection against ALA-induced mitochondrial transmembrane potential loss due to its highly liposoluble structure that can act directly on the mitochondrial membrane.⁴⁸ Supplemental antioxidants were able to inhibit ALA stimulatory effect on calcium ion uptake by 50%.⁴⁸ This is promising for those suffering from the neurodegenerative effects of lead poisoning, as limiting these effects of ALA accumulation can mitigate the mitochondrial impairment (see Figure 11).