Inhibition of Plasmodium falciparum triose-phosphate isomerase by chemical modification of an interface cysteine- electrospray ionization mass spectrometric analysis of differential cysteine reactivities

Plasmodium falciparumtriose-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 is > 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

Crosstalk between purine nucleotide metabolism and mitochondrial pathways in Plasmodium falciparum

Metabolism is known for its intricate adjustments to meet the needs of an organism. Due to demands of adaptation, parasite metabolic pathways are greatly altered from those of their hosts. A key difference in metabolic pathways in Plasmodium spp, during the intra-erythrocytic stages, pertains to energy metabolism with the absence of a major role for the mitochondria in adenosine triphosphate (ATP) generation. In most organisms there exists a tight link between nucleotide and energy metabolism. An important feature of purine nucleotide metabolism in Plasmodium falciparum is the absence of the de novo pathway, with purine requirements being completely met by the salvage pathway. Presence of the enzymes adenylosuccinate lyase, adenylosuccinate synthetase and adenosine monophosphate (AMP) deaminase, involved in AMP metabolism, suggests the existence of a functional purine nucleotide cycle (PNC) in P. falciparum with fumarate, a tricarboxylic acid (TCA) cycle intermediate, and ammonia being the net output from the cycle. In the absence of a conventional TCA cycle, the fate of fumarate generated from PNC merits examination. In this review we cover ATP generation through glycolysis, key features of the TCA cycle, the role of electron transport chain and the link between PNC and the mitochondrion. Recent studies using genetic approaches highlight unexpected features that were hitherto unknown with respect to these pathways and these are also summarized in this review

Unusual fluorescence of W168 in Plasmodium falciparum triosephosphate isomerase, probed by single-tryptophan mutants

Plasmodium falciparum triosephosphate isomerase (PfTIM) contains two tryptophan residues, W11 and W168. One is positioned in the interior of the protein, and the other is located on the active-site loop 6. Two single-tryptophan mutants, W11F and W168F, were constructed to evaluate the contributions of each chromophore to the fluorescence of the wild-type (wt) protein and to probe the utility of the residues as spectroscopic reporters. A comparative analysis of the fluorescence spectra of PfTIMwt and the two mutant proteins revealed that W168 possesses an unusual, blue-shifted emission (321 nm) and exhibits significant red-edge excitation shift of fluorescence. In contrast, W11 emits at 332 nm, displays no excitation dependence of fluorescence, and behaves like a normal buried chromophore. W168 has a much shorter mean lifetime (2.7 ns) than W11 (4.6 ns). The anomalous fluorescence properties of W168 are abolished on unfolding of the protein in guanidinium chloride (GdmCl) or at low pH. Analysis of the tryptophan environment using a 1.1-Å crystal structure established that W168 is rigidly held by a complex network of polar interactions including a strong hydrogen bond from Y164 to the indole NH group. The environment is almost completely polar, suggesting that electrostatic effects determine the unusually low emission wavelength of W168. To our knowledge this is a unique observation of a blue-shifted emission from a tryptophan in a polar environment in the protein. The wild-type and mutant proteins show similar levels of enzymatic activity and secondary and tertiary structure. However, the W11F mutation appreciably destabilizes the protein to unfolding by urea and GdmCl. The fluorescence of W168 is shown to be extremely sensitive to binding of the inhibitor, 2-phosphoglycolic acid

Proteolytic stability of β-peptide bonds probed using quenched fluorescent substrates incorporating a hemoglobin cleavage site

AbstractA set of designed internally quenched fluorescence peptide substrates has been used to probe the effects of insertion of β-peptide bonds into peptide sequences. The test sequence chosen corresponds to a proteolytically susceptible site in hemoglobin α-chain, residues 32–37. Fluorescence and mass spectral measurements demonstrate that the insertion of an β-residues at the potential cleavage sites completely abolishes the action of proteases; in addition, the rate of cleavage of the peptide bond preceding the site of modification is also considerably reduced

Cloning and characterization of the Plasmodium falciparum adenylosuccinate synthetase gene

The cloning and deduced amino acid sequence of Plasmodium falciparum adenylosuccinate synthetase (PfADSS) are reported. PfADSS exhibited 67% homology with the human enzyme. On expression in Escherichia coli, enzymatically active ADSS was produced as deduced by functional complementation analysis. The PfADSS activity was inhibited by hadacidin, a known competitive inhibitor of this enzyme. Nucleotide sequence data are available at GenBank under accession number AF095282

Synthetic peptide models for protein secondary structures β-sheet formation in acyclic cystine peptides

The conformations of the symmetrical cystine peptides Boc-Cys -(Val)<SUB>n</SUB> -Trp-OMe Boc-Cys-(Val)<SUB>n</SUB> -Trp-OMe (n = 1, 1; 2, 2; 3, 3) have been examined in solution, in order to evaluate the use of disulfide crosslinks in stabilizing extended &#946;-strand conformations in acyclic sequences. NMR studies in (CD<SUB>3</SUB>)<SUB>2</SUB> SO provide evidence for the solvent inaccessible nature of the Val (2) NH group in peptides 1 and 2. J<SUB>HNCHxH</SUB> values are indicative of extended structures. Sequential interresidue nuclear Overhauser effects support the population of &#946;-strand structures in both peptides. The fluorescence quantum yield of tryptophan determined in methanol follows the order 2 &gt; 1 n 3. Reduction of the disulfides with NaBH<SUB>4</SUB> results in large enhancements of emission intensity, with the changes following the order 1 &gt; 3 &gt;&gt; 2. The order of quenching is a function of the disposition of the indole and disulfide sidechains in an extended &#946; -sheet structure

Structural effects of a dimer interface mutation on catalytic activity of triosephosphate isomerase.The role of conserved residues and complementary mutations

The active site of triosephosphate isomerase (TIM, EC: 5.3.1.1), a dimeric enzyme, lies very close to the subunit interface. Attempts to engineer monomeric enzymes have yielded well-folded proteins with dramatically reduced activity. The role of dimer interface residues in the stability and activity of the Plasmodium falciparum enzyme, PfTIM, has been probed by analysis of mutational effects at residue 74. The PfTIM triple mutant W11F/W168F/Y74W (Y74W*) has been shown to dissociate at low protein concentrations, and exhibits considerably reduced stability in the presence of denaturants, urea and guanidinium chloride. The Y74W* mutant exhibits concentration-dependent activity, with an approximately 22-fold enhancement of kcat over a concentration range of 2.5–40 μm, suggesting that dimerization is obligatory for enzyme activity. The Y74W* mutant shows an approximately 20-fold reduction in activity compared to the control enzyme (PfTIM WT*, W11F/W168F). Careful inspection of the available crystal structures of the enzyme, together with 412 unique protein sequences, revealed the importance of conserved residues in the vicinity of the active site that serve to position the functional K12 residue. The network of key interactions spans the interacting subunits. The Y74W* mutation can perturb orientations of the active site residues, due to steric clashes with proximal aromatic residues in PfTIM. The available crystal structures of the enzyme from Giardia lamblia, which contains a Trp residue at the structurally equivalent position, establishes the need for complementary mutations and maintenance of weak interactions in order to accommodate the bulky side chain and preserve active site integrity

Stereochemistry of \alpha-Aminoisobutyric Acid Peptides in Solution: Conformations of Decapeptides with a Central Triplet of Contiguous L-Amino Acids

The decapeptides $Boc-Aib-L-Val-Aib-Aib-{(L-Val)}_3-Aib-L-Val-Aib-OMe$ and $Boc-Aib-L-Leu- Aib-Aib-{(L-Leu)}_3-Aib-L-Leu-Aib-OMe$ have been studied in $CD{Cl}_3$ and ${({CD}_3)}_2SO$ solutions by 270-MHz' H-nmr. In $CD{Cl}_3$ the presence of eight intramolecularly hydrogen-bonded NH groups has been established, consistent with a $3_{10}$-helical conformation, for both decapeptides. In ${({CD}_3)}_2SO$ only seven solvent-shielded NH groups are observed, supporting either an \alpha-helical conformation or a partially unfolded $3_{10}$-helix. Ir studies provided supporting evidence for intramolecularly hydrogen-bonded structures in $CH{Cl}_3$, while CD studies suggest helical conformation in both decapeptides in various solvents. CD studies also support helical folding in the C-terminal hexapeptides. The central triplet of L-amino acids appears to destabilize $3_{10}$-helical conformations in polar solvents like ${({CD}_3)}_2SO$

Structural effects of a dimer interface mutation on catalytic activity of triosephosphate isomerase.The role of conserved residues and complementary mutations

The active site of triosephosphate isomerase (TIM, EC: 5.3.1.1), a dimeric enzyme, lies very close to the subunit interface. Attempts to engineer monomeric enzymes have yielded well-folded proteins with dramatically reduced activity. The role of dimer interface residues in the stability and activity of the Plasmodium falciparum enzyme, PfTIM, has been probed by analysis of mutational effects at residue 74. The PfTIM triple mutant W11F/W168F/Y74W (Y74W*) has been shown to dissociate at low protein concentrations, and exhibits considerably reduced stability in the presence of denaturants, urea and guanidinium chloride. The Y74W* mutant exhibits concentration-dependent activity, with an approximately 22-fold enhancement of kcat over a concentration range of 2.5–40 μm, suggesting that dimerization is obligatory for enzyme activity. The Y74W* mutant shows an approximately 20-fold reduction in activity compared to the control enzyme (PfTIM WT*, W11F/W168F). Careful inspection of the available crystal structures of the enzyme, together with 412 unique protein sequences, revealed the importance of conserved residues in the vicinity of the active site that serve to position the functional K12 residue. The network of key interactions spans the interacting subunits. The Y74W* mutation can perturb orientations of the active site residues, due to steric clashes with proximal aromatic residues in PfTIM. The available crystal structures of the enzyme from Giardia lamblia, which contains a Trp residue at the structurally equivalent position, establishes the need for complementary mutations and maintenance of weak interactions in order to accommodate the bulky side chain and preserve active site integrity

Multiple Conformational States of a Pro-Pro Peptide. Solid-State and Solution Conformations of Boc-Aib-Pro-Pro-NHMe

The solid-state and solution conformations of the model peptide Boc-Aib-Pro-Pro-NHMe have been studied by X-ray diffraction and NMR. The peptide adopts a poly(pro1ine 11) conformation in the solid state. Two molecules are observed in the asymmetric unit differing in the geometry (cisltrans) of the urethane group. The molecules are held together in the crystal by a complex network of hydrogen bonds involving three molecules of water, which cocrystallize. Dissolution of single crystals at low temperature (\sim 233 K) permits NMR observation of the solid-state conformer. In solution, the peptide undergoes a trans-cis isomerization of the Pro-Pro bond. Low- temperature NMR measurements allow the detection of three conformational states of the Pro-Pro segment. Both cis’ and trans’ rotational isomers about the $C^\alpha -CO$ $(\psi)$ bond of Pro-3 are detectable at low temperatures. Theoretical calculations suggest an appreciable activation barrier to $\psi$ rotation. Temperature and solvent dependence of NH chemical shifts provide evidence for an intramolecular hydrogen bond, involving the NHMe group in the cis Pro-Pro conformer. Energy calculations suggest the possibility of a type VI $\beta$-turn conformation stabilized by a 4 $\rightarrow$ 1 hydrogen bond between the Aib-1 CO and NHMe groups