Inhibition of plasmodium falciparum triose-phosphate isomerase by chemical modification of an interface cysteine. Electrospray ionization mass spectrometric analysis of differential cysteine reactivities.
Plasmodium falciparum triose-phosphate isomerase, a homodimeric enzyme, contains four cysteine residues at positions 13, 126, 196, and 217 per subunit. Among these, Cys-13 is present at the dimer interface and is replaced by methionine in the corresponding human enzyme. We have investigated the effect of sulfhydryl labeling on the parasite enzyme, with a view toward developing selective covalent inhibitors by targeting the interface cysteine residue. Differential labeling of the cysteine residues by iodoacetic acid and iodoacetamide has been followed by electrospray ionization mass spectrometry and positions of the labels determined by analysis of tryptic fragments. The rates of labeling follows the order Cys-196 > Cys-13 Cys-217/Cys-126, which correlates well with surface accessibility calculations based on the enzyme crystal structure. Iodoacetic acid labeling leads to a soluble, largely inactive enzyme, whereas IAM labeling leads to precipitation. Carboxyl methylation of Cys-13 results in formation of monomeric species detectable by gel filtration. Studies with an engineered C13D mutant permitted elucidation of the effects of introducing a negative charge at the interface. The C13D mutant exhibits a reduced stability to denaturants and 7-fold reduction in the enzymatic activity even under the concentrations in which dimeric species are observed.
Maithal
Kapil
K
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India.
Ravindra
Gudihal
G
Balaram
Hemalatha
H
Balaram
Padmanabhan
P
D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000737:chemistry*; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010963:Plasmodium falciparum / Q000201:enzymology*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization; D014305:Triose-Phosphate Isomerase / Q000737:chemistry / Q000302:isolation & purification*
D014305:Triose-Phosphate Isomerase; D003545:Cysteine