1. Biochemistry of Plasmodium
Mark F. Wiser

2. Today I will be talking about the biochemistry of the malaria parasite.
Biochemistry is the study of the interconversion of molecules as depicted by this figure with each dot representing a distinct chemical and the lines representing enzymes that are responsible for the conversions
All organisms are made of macromolecules. These macromolecules are composed of subunits. (Table)
Living and growing organisms require these building blocks. Nutrients are acquired from the environment and converted into substances or energy needed by the organism.
Catabolism is destructive. Anabolism is constructive
Why study biochemistry of Plasmodium? It will be similar to other organisms. This question will be the focus of the second lecture on drug action. Namely, biochemical differences can be exploited.
For example, a pathogen may have unique pathways not found in the host. Or a pathway may be more important in the pathogen than the host. Or the pathway is equally important, but drugs specific for enzymes of the pathogen are available.
Parasite has huge demand for precursors, especially DNA. Antifolates block synthesis of nucleotides and inhibit the parasite
Today will focus on proteins and amino acids. Central importance and many known antimalarials affect protein metabolism.

3. Sources of Amino Acids De Novo Synthesis Host Plasma
CO2 fixation (ala, asp, glu)
little incorporated into protein
Host Plasma
 uptake of all amino acids
in vitro growth requires ile, met, cys, gln, glu
Digestion of Host Hemoglobin
16% of amino acids derived from hemoglobin are incorporated into parasite proteins

4. Hemoglobin 95% of total erythrocyte protein
very abundant (340 mg/ml or approximately 5 mM)
60-80% is degraded during erythrocytic stage
110 g (of 750 total) is consumed in 48 hrs at 20% parasitemia
Hb is major protein in erythrocyte
Parasite degrades a large proportion.
Host mass is converted into parasite mass.
How is this accomplished? …..

5. Endocytosis of Host Cytoplasm
cytostome
food vacuole
An early step in the digestion of Hb is its endocytocis
pinocytosis during ring stage
specialized organelle called cytostome in later stages
PVM membrane is broken down
Hemoglobin is digested within vacuole called food vacuole
Late in trophozoite stage the small food vacuoles coalesce into large food vacuole
malaria pigment, or hemozoin, is the waste product from Hb digestion
What is this FV? ….
pinocytosis (rings)

6. The Food Vacuole A Specialized Lysosome
ATP
hemoglobin digestion H+
(pH 5-5.4)
ADP
Food Vacuole Proteases
plasmepsins I - IV (acid)
falcipains I - III (thiol)
falcilysin (metallo)
Absent:
other acid hydrolases except acid phosphatase
Endocytic
Pathway
parasite
cytoplasm
Several distinct proteases have been identified in FV
Fv is analogous to lysosome
acid compartment with hydrolases
part of endocytic pathway
noted differences are lack of other hydrolases (not needed because of erythrocyte)
proteases are enzymes that break down proteins into peptides or amino acids
this can be illustrated in the following …

7. Proteases Mediate the Catabolism of Proteins
proteases (aka peptidases) break the peptide bonds that hold amino acids together
exopeptidases remove amino acids sequentially from either N- or C-terminus
endopeptidases cleave between ‘specific’ residues within polypeptide chain
Proteases are enzymes that hydrolyze peptide bonds
endo vs. exo
What about the proteases in food vacuole?
all are endo
lets look at these proteases in regards to the digestion of Hb ….

8. Initial plasmepsin cleavage is specific and leads to a destabilization of hemoglobin
native Hb is cleaved between Phe-33 and Leu-34 ( chains)
‘hinge region’
conserved
important for tetramer stability
the large globin fragments dissociate
heme is released
globin fragments are susceptible to further proteolysis
a-F33/L34 í
Native Hb is a globular protein made of 4 subunits--relative resistance to proteolysis
specific cleavage of Hb in hinge region results in protein falling apart
This will expose more protease sites

9. Hemoglobin Digestion is an Ordered Process
aminopeptidase
hemoglobin +
heme
large globin fragments
small peptides
(6-8 amino acids)
plasmepsin
falcipain
medium fragments
(~20 amino acids)
falcilysin
amino acids
di-peptides
dipeptidyl aminopeptidase
The release of the globin fragments exposes more protease sites for the continued digestion of the polypeptides
falcipains and plasmepsins will act at numerous places (globin chain approximately. 140 residues)
falcilysin does not efficiently digest fragment larger that 20 residues. Probably responsible for going down to fragments of 6-8 residues
These peptides are then further degraded to amino acids
However, not complete due to specificity of proteases
Final result is mixture of amino acids and peptides
So how do these get exported into host cytoplasm where they are need for further metabolism and protein synthesis? ...
End-products are small peptides, dipeptides, and amino acids

10. Membrane Transport
Hydrophilic solutes require transporters (ie, carrier proteins)
substrate specific
most require energy
ATPase or gradients (eg, H+)
6 amino acid transporters identified in Plasmodium genome (location?)
PfMDR-1 and PfCRT located on food vacuole membrane
Two ways in which small molecules are translocted across membranes: channels and carriers
For amino acids carriers are most important
Generally substrate specific and require energy
In the genome there are 6 potential amino acid transporters, but none have been further characterized
ABC transporters are also important ....

11. PfMDR-1 Member of ABC (ATP-binding cassette) transporter super family
Associated with drug resistance (MDR = multi-drug resistance)
Capable of peptide transport
complements yeast ste6 gene (transporter of yeast peptide mating factor)
However, PfMDR-1 imports solutes (including drugs) into food vacuole

12. PfCRT Member of DMT (drug/metabolite transporter) super family
H+-coupled polyspecific nutrient exporter including amino acids and peptides
Associated with chloroquine resistance (CRT = chloroquine resistance transporter)
identified through genetic linkage studies
Exports chloroquine and other drugs from the food vacuole

13. heme is by-product of hemoglobin catabolism
PfCRT is probable transporter
amino-peptidase activity in cytoplasm

14. Free Heme is Toxic heme destabilizes and lyses membranes parasite dies
hydrolytic enzymes released into parasite cytoplasm
parasite dies
Possible Detoxification Mechanisms
heme  hemozoin (malaria pigment)
H2O2 mediated degradation
GSH mediated degradation
heme oxygenase (P.b. and P.k. only)
Free heme is toxic
destabilizes membranes leading to cell death
detoxified by several possible mechanisms—predominant is formation of hemozoin or pigment
Polymerized heme is the malarial pigment (=hemazoin)
waste product that is encapsulated in residual body after merozite budding. Found deposited in the tissues--well known phenomenon
what is hemazoin?

15. Hemozoin = b-Hematin heme b-hematin dimer formation
X-ray crystallography and spectroscopic analysis indicates that hemozoin has the same structure as b-hematin
b-hematin is a heme dimer formed via reciprocal covalent bonds between carboxylic acid groups on the protoporphyrin-IX ring and the iron atoms of two heme molecules
heme
b-hematin

16. b-hematin forms insoluble crystals
biocrystallization
These dimers interact through hydrogen bonds to form crystals of hemozoin.
Therefore, pigment formation is best described as a biocrystalization, or biomineralization, process
hemozoin can form spontaneously under harsh conditions (non-biological)

17. Pigment Formation biocrystallization mechanism unknown
lipid bodies can promote the process
possibly derived from PVM
potential heme detoxification protein (HDP)
unique to Plasmodium species
exported to host cytoplasm and taken up into food vacuole
binds 2 heme groups with high affinity (80 nM) promoting b-hematin dimer formation
dimer seeds biocrystallization reaction?
heme biocrystallization inhibited by chloroquine and other anti-malarials
Hemozoin can be formed chemically. But under harsh conditions. Parasite proteins likely involved. But none identified.
Histidine rich proteins and lipids have been implicated.
Polymerization inhibited by CQ and other 4-aminoquinolines. CQ kills by build of toxic free heme.
The updated Fv now looks like this ...

18. The Food Vacuole A Specialized Lysosome
ATP
hemoglobin H+
Fe2+
plasmepsin O2
globin
fragments
ADP
Fe3+
heme +
amino
acids
-O2 O2
-lipids?
-detox protein?
multiple
proteases
iron oxidized after release from Hb
oxidation promotes formation of ROS
oxidative stress
The release of free heme also leads to ROI which place additional stress on the parasite.
hemozoin
amino-
peptidase
small peptides
di-amino acids
amino acids
PfCRT H+

19. The Food Vacuole A Specialized Lysosome
ATP
hemoglobin H+
Fe2+
plasmepsin O2
globin
fragments
ADP
Fe3+
heme +
amino
acids
-O2 O2
-lipids?
-detox protein?
superoxide
dismutase?
multiple
proteases
SOD and catalase, possibly derived from host may help protect against oxygen stress.
Some genetic conditions place additional oxygen stress as well as some antimalarials.
Summary:
H2O2
hemozoin
amino-
peptidase
catalase?
peroxidase?
small peptides
di-amino acids
amino acids
PfCRT
H2O + O2 H+
parasite has SOD and peroxidases, but no catalase
location?

20. Summary Food vacuole is lysosome-like organelle
Digestion of hemoglobin and detoxification of heme are primary metabolic activities
Crucial organelle for the parasite and provides many potential drug targets