Structural Insights into the Molecular Design of Flutolanil Derivatives Targeted for Fumarate Respiration of Parasite Mitochondria.

Recent studies on the respiratory chain of Ascaris suum showed that the mitochondrial NADH-fumarate reductase system composed of complex I, rhodoquinone and complex II plays an important role in the anaerobic energy metabolism of adult A. suum. The system is the major pathway of energy metabolism for adaptation to a hypoxic environment not only in parasitic organisms, but also in some types of human cancer cells. Thus, enzymes of the pathway are potential targets for chemotherapy. We found that flutolanil is an excellent inhibitor for A. suum complex II (IC50 = 0.058 µM) but less effectively inhibits homologous porcine complex II (IC50 = 45.9 µM). In order to account for the specificity of flutolanil to A. suum complex II from the standpoint of structural biology, we determined the crystal structures of A. suum and porcine complex IIs binding flutolanil and its derivative compounds. The structures clearly demonstrated key interactions responsible for its high specificity to A. suum complex II and enabled us to find analogue compounds, which surpass flutolanil in both potency and specificity to A. suum complex II. Structures of complex IIs binding these compounds will be helpful to accelerate structure-based drug design targeted for complex IIs.

Peptidases compartmentalized to the Ascaris suum intestinal lumen and apical intestinal membrane.

The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine.

Molecular cloning and analysis of Ancylostoma ceylanicum glutamate-cysteine ligase.

Glutamate-cysteine ligase (GCL) is a heterodimer enzyme composed of a catalytic subunit (GCLC) and a modifier subunit (GCLM). This enzyme catalyses the synthesis of γ-glutamylcysteine, a precursor of glutathione. cDNAs of the putative glutamate-cysteine ligase catalytic (Ace-GCLC) and modifier subunits (Ace-GCLM) of Ancylostoma ceylanicum were cloned using the RACE-PCR amplification method. The Ace-gclc and Ace-gclm cDNAs encode proteins with 655 and 254 amino acids and calculated molecular masses of 74.76 and 28.51kDa, respectively. The Ace-GCLC amino acid sequence shares about 70% identity and 80% sequence similarity with orthologs in Loa loa, Onchocerca volvulus, Brugia malayi, and Ascaris suum, whereas the Ace-GCLM amino acid sequence has only about 30% sequence identity and 50% similarity to homologous proteins in those species. Real-time PCR analysis of mRNA expression in L3, serum stimulated L3 and adult stages of A. ceylanicum showed the highest level of Ace-GCLC and Ace-GCLM expression occurred in adult worms. No differences were detected among adult hookworms harvested 21 and 35dpi indicating expression of Ace-gclc and Ace-gclm in adult worms is constant during the course of infection. Positive interaction between two subunits of glutamate-cysteine ligase was detected using the yeast two-hybrid system, and by specific enzymatic reaction. Ace-GCL is an intracellular enzyme and is not exposed to the host immune system. Thus, as expected, we did not detect IgG antibodies against Ace-GCLC or Ace-GCLM on days 21, 60 and 120 of A. ceylanicum infection in hamsters. Furthermore, vaccination with one or both antigens did not reduce worm burdens, and resulted in no improvement of clinical parameters (hematocrit and hemoglobin) of infected hamsters. Therefore, due to the significant role of the enzyme in parasite metabolism, our analyses raises hope for the development of a successful new drug against ancylostomiasis based on the specific GCL inhibitor.

Ascaris suum enolase is a potential vaccine candidate against ascariasis.

Ascariasis caused by Ascaris is the most common parasite problem in humans and pigs worldwide. No vaccines are available for the prevention of Ascaris infections. In the present study, the gene encoding Ascaris suum enolase (As-enol-1) was amplified, cloned and sequenced. Amino acid sequence alignment indicated that As-enol-1 was highly conserved between different nematodes and shared the highest identity (87%) with enolase from Anisakis simplex s.l. The recombinant pVAX-Enol was successfully expressed in Marc-145 cells. The ability of the pVAX-Enol for inducing immune protective responses against challenge infection with A. suum L3 was evaluated in Kunming mice. The immune response was evaluated by lymphoproliferative assay, cytokine and antibody measurements, and the reduction rate of recovery larvae. The results showed that the mice immunized with pVAX-Enol developed a high level of specific antibody responses against A. suum, a strong lymphoproliferative response, and significant levels of IFN-γ, IL-2, IL-4 and IL-10 production, compared with the other groups immunized with empty plasmid or blank controls, respectively. There was a 61.13% reduction (P<0.05) in larvae recovery compared with that in the blank control group. Our data indicated that A. suum enolase is a potential vaccine candidate against A. suum infection.

Role of residues in the adenosine binding site of NAD of the Ascaris suum malic enzyme.

Ascaris suum mitochondrial malic enzyme catalyzes the divalent metal ion dependent conversion of l-malate to pyruvate and CO(2), with concomitant reduction of NAD(P) to NAD(P)H. In this study, some of the residues that form the adenosine binding site of NAD were mutated to determine their role in binding of the cofactor and/or catalysis. D361, which is completely conserved among species, is located in the dinucleotide-binding Rossmann fold and makes a salt bridge with R370, which is also highly conserved. D361 was mutated to E, A and N. R370 was mutated to K and A. D361E and A mutant enzymes were inactive, likely a result of the increase in the volume in the case of the D361E mutant enzyme that caused clashes with the surrounding residues, and loss of the ionic interaction between D361 and R370, for D361A. Although the K(m) for the substrates and isotope effect values did not show significant changes for the D361N mutant enzyme, V/E(t) decreased by 1400-fold. Data suggested the nonproductive binding of the cofactor, giving a low fraction of active enzyme. The R370K mutant enzyme did not show any significant changes in the kinetic parameters, while the R370A mutant enzyme gave a slight change in V/E(t), contrary to expectations. Overall, results suggest that the salt bridge between D361 and R370 is important for maintaining the productive conformation of the NAD binding site. Mutation of residues involved leads to nonproductive binding of NAD. The interaction stabilizes one of the Rossmann fold loops that NAD binds. Mutation of H377 to lysine, which is conserved in NADP-specific malic enzymes and proposed to be a cofactor specificity determinant, did not cause a shift in cofactor specificity of the Ascaris malic enzyme from NAD to NADP. However, it is confirmed that this residue is an important second layer residue that affects the packing of the first layer residues that directly interact with the cofactor.

Optimum activity of the phosphofructokinase from Ascaris suum requires more than one metal ion.

Phosphofructokinase (PFK) catalyzes the phosphorylation of fructose 6-phosphate (F6P) to give fructose 1,6-bisphosphate (FBP) using MgATP as the phosphoryl donor. As the concentration of Mg(2+) increases above the concentration needed to generate the MgATP chelate complex, a 15-fold increase in the initial rate was observed at low MgATP. The effect of Mg(2+) is limited to V/K(MgATP), and initial rate studies indicate an equilibrium-ordered addition of Mg(2+) before MgATP. Isotope partitioning of the dPFK:MgATP complex indicates a random addition of MgATP and F6P at low Mg(2+), with the rate of release of MgATP from the central E:MgATP:F6P complex 4-fold faster than the net rate constant for catalysis. This can be contrasted with the ordered addition of MgATP prior to F6P at high Mg(2+). The addition of fructose 2,6-bisphosphate (F26P(2)) has no effect on the mechanism at low Mg(2+), with the exception of a 4-fold increase in the affinity of the enzyme for F6P. At high Mg(2+), F26P(2) causes the kinetic mechanism to become random with respect to MgATP and F6P and with MgATP released from the central complex half as fast as the net rate constant for catalysis. The latter is in agreement with previous studies [Gibson, G. E., Harris, B. G., and Cook, P. F. (1996) Biochemistry 35, 5451-5457]. The overall effect of Mg(2+) is a decrease in the rate of release of MgATP from the E:MgATP:F6P complex, independent of the concentration of F26P(2).

Role of dihydrolipoyl dehydrogenase (E3) and a novel E3-binding protein in the NADH sensitivity of the pyruvate dehydrogenase complex from anaerobic mitochondria of the parasitic nematode, Ascaris suum.

The pyruvate dehydrogenase complex (PDC) plays changing roles during the aerobic-anaerobic transition in the life cycle of the parasitic nematode, Ascaris suum. However, the dihydrolipoyl dehydrogenase (E3) subunit appears to be identical in all stages, despite the fact that the PDC is less sensitive to NADH inhibition in anaerobic muscle. Therefore, we have cloned cDNAs encoding E3 and a novel anaerobic-specific E3-binding protein (E3BP) that lacks the terminal lipoyl domain found in E3BPs from yeast and mammals, and functionally expressed E3 and E3 mutants designed to have decreased dimer stability on the assumption that the binding of E3 to an anaerobic-specific E3BP might stabilize the E3 dimer interface and decrease E3 sensitivity to NADH inhibition. As predicted, the mutants exhibited decreased thermal stability, increased sensitivity to NADH and the binding of E3(Y18F) to the E3-depleted core of the pig heart PDC increased E3 activity and decreased E3 sensitivity to NADH inhibition. However, although the free A. suum E3 was less sensitive to NADH inhibition than the pig heart E3, both E3s were significantly more sensitive to NADH inhibition when assayed with dihydrolipoamide than their corresponding PDCs assayed with pyruvate. More importantly, the binding of rE3 to its core complex had little effect on its apparent K(m) for NAD(+), K(i) for NADH inhibition, or the NADH/NAD(+) ratio yielding 50% inhibition. These data suggest that although binding to the core stabilizes the E3 dimer interface, it does not play a significant role in reducing the sensitivity of the A. suum PDC to NADH inhibition during anaerobiosis.

Role of cysteine 306 in the catalytic mechanism of Ascaris suum phosphoenolpyruvate carboxykinase.

Biochemical and metabolic data lead to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. The Ascaris suum PEPCK shares considerable homology with PEPCK from avian liver and is a good candidate for mutagenesis studies. The Cys306 substitution by Ser and Ala produced active enzymes and the two mutants are kinetically indistinguishable from each other. This substitution affects the catalytic affinity for the formation of the specific enzyme-nucleotide complex (k(cat)/K(m)) in the forward and reverse reactions. Studies with the substrate analogs 2(')dGDP and 2(')dGTP indicate that Cys306 in A. suum PEPCK is one of the residues important in nucleotide binding and may interact with the 2(')OH group in the ribose ring. Alternatively, mutation of this residue could cause protein changes that interfere with the proper conformation of the nucleotides for optimal catalysis to take place.

Stage-specific isoforms of Ascaris suum complex. II: The fumarate reductase of the parasitic adult and the succinate dehydrogenase of free-living larvae share a common iron-sulfur subunit.

Complex II of adult Ascaris suum muscle exhibits high fumarate reductase (FRD) activity and plays a key role in anaerobic electron-transport during adaptation to their microaerobic habitat. In contrast, larval (L2) complex II shows a much lower FRD activity than the adult enzyme, and functions as succinate dehydrogenase (SDH) in aerobic respiration. We have reported the stage-specific isoforms of complex II in A. suum mitochondria, and showed that at least the flavoprotein subunit (Fp) and the small subunit of cytochrome b (cybS) of the larval complex II differ from those of adult. In the present study, complete cDNAs for the iron-sulfur subunit (Ip) of complex II, which with Fp forms the catalytic portion of complex II, have been cloned and sequenced from anaerobic adult A. suum, and the free-living nematode, Caenorhabditis elegans. The amino acid sequences of the Ip subunits of these two nematodes are similar, particularly around the three cysteine-rich regions that are thought to comprise the iron-sulfur clusters of the enzyme. The Ip from A. suum larvae was also characterized because Northern hybridization showed that the adult Ip is also expressed in L2. The Ip of larval complex II was recognized by the antibody against adult Ip, and was indistinguishable from the adult Ip by peptide mapping. The N-terminal 42 amino acid sequence of Ip in the larval complex II purified by DEAE-cellulofine column chromatography was identical to that of the mature form of the adult Ip. Furthermore, the amino acid composition of larval Ip determined by micro-analysis on a PVDF membrane is almost the same as that of adult Ip. These results, together with the fact, that homology probing by RT-PCR, using degenerated primers, failed to find a larval-specific Ip, suggest that the two different stage-specific forms of the A. suum complex II share a common Ip subunit, even though the adult enzyme functions as a FRD, while larval enzyme acts as an SDH.

Kinetic characterization of a T-state of Ascaris suum phosphofructokinase with heterotropic negative cooperativity by ATP eliminated.

The affinity analogue, 2',3'-dialdehyde ATP has been used to chemically modify the ATP-inhibitory site of Ascaris suum phosphofructokinase, thereby locking the enzyme into a less active T-state. This enzyme form has a maximum velocity that is 10% that of the native enzyme in the direction of fructose 6-phosphate (F6P) phosphorylation. The enzyme displays sigmoid saturation for the substrate fructose 6-phosphate (S0.5 (F6P) = 19 mM and nH = 2.2) at pH 6.8 and a hyperbolic saturation curve for MgATP with a Km identical to that for the native enzyme. The allosteric effectors, fructose 2,6-bisphosphate and AMP, do not affect the S0.5 for F6P but produce a slight (1.5- and 2-fold, respectively) V-type activation with Ka values (effector concentration required for half-maximal activation) of 0.40 and 0.24 mM, respectively. Their activating effects are additive and not synergistic. The kinetic mechanism for the modified enzyme is steady-state-ordered with MgATP as the first substrate and MgADP as the last product to be released from the enzyme surface. The decrease in V and V/K values for the reactants likely results from a decrease in the equilibrium constant for the isomerization of the E:MgATP binary complex, thus favoring an unisomerized form. The V and V/KF6P are pH dependent with similar pK values of about 7 on the acid side and 9.8 on the basic side. The microenvironment of the active site appears to be affected minimally as evidenced by the similarity of the pK values for the groups involved in the binding site for F6P in the modified and native enzymes.

Effect of protease class-specific inhibitors on in vitro development of the third- to fourth-stage larvae of Ascaris suum.

Third-stage larvae (L3) of Ascaris suum develop and molt to fourth-stage larvae (L4) during in vitro cultivation; consistently greater than 80% of the larvae develop to L4 during 7 days in culture (DIC). To assess the role of proteases in this process, the effect of protease class-specific inhibitors was studied. The presence of either a serine protease inhibitor (AEBSF, 100 microM) or an aspartic protease inhibitor (pepstatin A, 100 microM) had no effect on the percentage of L4 after 7 DIC. However, the presence of either a cysteine protease inhibitor (Z-Phe-Ala-FMK, 100 microM) or an aminopeptidase inhibitor (amastatin, 100 microM) resulted in 77% and 34% reductions, respectively, in the percentage of L4 compared to untreated cultures; viability of the larvae was not affected. The effect of Z-Phe-Ala-FMK on molting was time and dose dependent. In contrast to Z-Phe-Ala-FMK, E-64, another specific inhibitor of cysteine proteases, had no effect on molting. The data support a role for an aminopeptidase and suggest a role for a cysteine protease in the development of the L3 to L4 stage of A. suum.

Purification and properties of a membrane aminopeptidase from Ascaris suum muscle that degrades neuropeptides AF1 and AF2.

We have identified on the membranes of the locomotory muscle of Ascaris suum an amastatin-sensitive aminopeptidase that hydrolyses the bioactive neuropeptides AF1 (KNEFIRF-NH2) and AF2 (KHEYLRF-NH2), by cleavage of the Lys1-Asn2 and Lys1-His2 peptide bonds, respectively. AF2 (1.2 nmol of HEYLRF-NH2 formed min[-1] (mg protein[-1])) was hydrolysed at a faster rate compared to AF1 (0.2 nmol of NEFIRF-NH2 formed min[-1] (mg protein[-1])). AF1 hydrolysis by the aminopeptidase was inhibited by the amastatin (IC50, 9.0 microM), leuhistin (IC50, 1.25 microM) but was insensitive to puromycin, indicating a similarity to mammalian aminopeptidase N. The enzyme was also inhibited by arphamenine B (IC50, 9.0 microM), (2S, 3R)-3-amino-2-hydroxy-4-(4-nitrophenyl)butanoyl-L-leucine (IC50, 8.0 microM), bestatin (IC50, 15.0 microM) and 1 mM 1-10 bis-phenanthroline. The detergent Triton X-100 solubilised enzyme had a pI of 5.0 and after 1000-fold purification by ion-exchange chromatography, appeared to have a Mr of around 240,000 by SDS-PAGE. The purified aminopeptidase had a Km of 534 microM for the hydrolysis of AF1 and cleaved Phe1 from FMRF-NH2, but was unable to hydrolyse DFMRF-NH2 or FDMRF-NH2. The aminopeptidase that we have described in this report might have a role in the extracellular metabolism and inactivation of neuropeptides acting on the locomotory muscle of A. suum.

Secretion of an aminopeptidase during transition of third- to fourth-stage larvae of Ascaris suum.

Protease activity was identified in culture fluids collected during in vitro development of L3 to L4 larval stages of Ascaris suum. Fluorogenic peptide substrates with unblocked N-termini were specifically hydrolyzed indicating aminopeptidase activity; a terminal arginyl residue was preferred. Culture fluids did not hydrolyze fluorogenic peptide substrates with blocked N-termini (endopeptidase substrates). The aminopeptidase activity was inhibited by 1,10-phenanthroline (metalloprotease inhibitor) and by amastatin and bestatin (aminopeptidase inhibitors); AEBSF (serine protease inhibitor), Z-phe-ala-FMK and E-64 (cysteine protease inhibitors), and pepstatin A (aspartyl protease inhibitor) had little effect on activity. The apparent molecular weight of the aminopeptidase was estimated by sucrose density gradient centrifugation at 293 kDa. The aminopeptidase displayed an acidic isoelectric point of 4.7. The peak secretion of the aminopeptidase was temporally associated with molting and suggests a function for the protease in this complex process.

Isotope partitioning with Ascaris suum phosphofructokinase is consistent with an ordered kinetic mechanism.

Isotope partitioning and initial velocity studies have been used to study the kinetic mechanism of Ascaris suum phosphofructokinase (PFK) at pH 8.0 for the native enzyme (nPFK), and at pH 6.8 for a form of enzyme desensitized (dPFK) to hysteresis in the reaction time course, to ATP allosteric inhibition, and to F6P homotropic cooperativity. Complete trapping (P*max approximately equal to 100%) of the E:MgATP* complex as fructose (1-32P)-1, 6-bisophosphate for both enzyme forms is consistent with the previously proposed steady-state ordered mechanism [Rao, G.S.J., Harris, B.G., & Cook, P.F. (1987) J.Biol. Chem. 262, 14074-14079] with MgATP binding before fructose 6-phosphate (F6P). K'F6P values for trapping of MgATP of 0.54 +/- 0.09 mM for nPFK and 0.85 +/- 0.15 mM for dPFK were obtained. Saturating amounts of the heterotropic activator fructose 2, 6-bisphosphate (F26P2) gives no change in the trapping parameters for nPFK with a P*max of 100% and a K'F6P of 0.40 +/- 0.06 mM. For dPFK, however, F26P2 causes a decrease in both parameters, giving a P*max of 54% and a K'F6P of 0.26 +/- 0.07 mM. The partial trapping of E:MgATP* in the presence of F26P2 for dPFK suggests that the activator changes the kinetic mechanism from an ordered to a random binding of substrates. Initial velocity studies confirm the change in mechanism. Uncompetitive inhibition by arabinose 5-phosphate (Ara5P), a dead-end inhibitory analog of F6P, versus MgATP for nPFK in the absence and presence of F26P2 is consistent with an ordered mechanism with MgATP adding to enzyme prior to F6P. An uncompetitive pattern is also obtained with dPFK for Ara5P versus MgATP in the absence of F26P2, but the pattern becomes noncompetitive in the presence of F26P2, consistent with a change to a random mechanism. No trapping of the E:[14C]F6P complex could be detected, indicating either that the E:[14C]F6P complex does not form in a significant amount under the conditions used or that the off-rate for F6P from enzyme is much faster than the net rate constant for formation of the first product, FBP. The data are consistent with a predominantly ordered mechanism with MgATP binding prior to F6P. The minor pathway with MgATP dissociating from the E:F6P:MgATP ternary complex becomes apparent for the dPFK in the presence of F26P2.

Comparison of the substrate specificities of cAMP-dependent protein kinase from bovine heart and Ascaris suum muscle.

The catalytic subunits of cAMP-dependent protein kinases (protein kinase A) from bovine heart and Ascaris suum muscle exhibit only 48% sequence identity and show quantitative differences in substrate specificity. These differences were not obvious at the level of short synthetic substrate peptides but were distinct for some protein substrates. Phosphofructokinase from Ascaris, a physiological substrate, was a better substrate for the protein kinase from the nematode in comparison to the mammalian protein kinase due to a 10-fold lower Michaelis constant. Selective phosphorylation by the two kinases was also observed with some in vitro substrates. In addition, quantitative differences in the interactions between R- and C-subunits from Ascaris and bovine heart were observed. However, several synthetic peptides whose sequence reflected the phosphorylation site of Ascaris suum phosphofructokinase (AKGRSDS*IV), or variations of it, were phosphorylated with the same efficiency by both protein kinases. Based on the data the following are concluded: (1) In agreement with the conservation of structure in the catalytic cleft, the recognition of substrates by protein kinases from phylogenetically distant organisms exhibits similarity. (2) Non-conserved parts of the surface of the protein kinase molecule may contribute to binding of protein substrates and thus to selective recognition.

Metabolism of AF1 (KNEFIRF-NH2) in the nematode, Ascaris suum, by aminopeptidase, endopeptidase and deamidase enzymes.

We have studied the metabolism and inactivation of AF1 (KNEFIRF-NH2) by membranes prepared from the locomotory muscle of Ascaris suum. FIRF-NH2 and KNEFIRF were identified as three primary degradation products, resulting from the action of an endopeptidase, aminopeptidase and a deamidase, respectively. The endopeptidase resembled mammalian neprilysin (NEP, endopeptidase 24.11) in that the enzyme activity was inhibited by phosphoramidon and thiorphan and that it cleaved AF1 on the amino side of phenylalanine. The aminopeptidase activity was inhibited by amastatin and bestatin but not by puromycin. The deamidation of AF1 was inhibited by phenylmethylsulfonyl fluoride, p-chloromercuricphenylsulfonate and mercuric chloride, indicating that the deamidase enzyme is a serine protease with a requirement for a free thiol group for activity. AF1 (1 microM) induces an increase in tension and an increase in the frequency and amplitude of spontaneous contractions of an A. suum muscle strip. None of the aforementioned AF1 metabolites (2-20 microM) retained biological activity in this bioassay, indicating that the endopeptidase, aminopeptidase and deamidase have the potential to terminate the action of AF1 on locomotory muscle of A. suum.

A pH-dependent allosteric transition in Ascaris suum phosphofructokinase distinct from that observed with fructose 2,6-bisphosphate.

Ascaris suum phosphofructokinase exhibits dramatic shifts in its circular dichroic spectra in the pH range 6 to 8. These shifts are quite distinct from those induced by the activators AMP and fructose 2,6-bisphosphate. Concomitant with these pH-induced spectral shifts, the enzyme also displays changes in its allosteric behavior. Inorganic ions such as K+, NH+4, SO4(2-), and PO4(3-) also cause CD-spectral shifts similar to those produced by a change in pH. Based on the evidence derived from gel filtration and sedimentation equilibrium studies, the observed CD-spectral shifts are interpreted as due to conformational changes in the enzyme tetramer rather than due to a change in its aggregation state. Further, since the pK value of 6.4 obtained from pH dependence of increase in ellipticity at 210 nm agrees very well with the pK value of 6.8 for the loss of ATP inhibition due to modification of a histidine residue (G. S. J. Rao, B. A. Wariso, P. F. Cook, and B. G. Harris (1987) J. Biol. Chem. 262, 14068-14073), it is concluded that a single histidine residue in the ATP-inhibitory site acts as a trigger for the structural changes accompanying ATP inhibition of the enzyme. This view is strongly supported by the observation that the enzyme desensitized to ATP inhibition by chemical modification of a histidine residue in the ATP-inhibitory site shows absolutely no change in its CD spectrum in the pH range 6 to 8. This study demonstrates that the mechanism of activation of phosphofructokinase at higher pH and by inorganic ions involves conformational transitions that are quite distinct from those induced by AMP and fructose 2,6-bisphosphate. A scheme is presented that incorporates all of the different states of the enzyme dependent upon effectors and pH.

The catalytic subunit of cAMP-dependent protein kinase from Ascaris suum. The cloning and structure of a novel subtype of protein kinase A.

A complete cDNA clone encoding the catalytic subunit of cAMP-dependent protein kinase of Ascaris suum was constructed from two overlapping partial clones. The encoded sequence of 337 amino acids is 48% identical with the sequence of mouse C alpha subunit. Approximately the same low similarity was found with the sequence of the C subunit from another nematode, Caenorhabditis elegans. The N-terminal 14 amino acids and the myristoylation site of the mammalian protein are not contained in the enzyme from Ascaris. Two cysteines (Cys33 and Cys319) replace a basic residue in the N-terminal region and an acidic amino acid near the C-terminus which are conserved in all known C subunits from other sources. The substitutions provide the possibility of disulfide bridge formation between the N-terminal and C-terminal parts of the protein. There is strong evidence that a single gene encodes cAMP-dependent protein kinase in Ascaris. Modelling of the sequence into the coordinates of the X-ray structure of the mammalian enzyme suggest a high degree of conservation in the three-dimensional structure. However, structural variations occur at the surface of the protein near the catalytic cleft and are likely to account for the variations in substrate specificity previously observed between the purified protein kinase from Ascaris [Thalhofer, H. P., Daum, G., Harris, B. G. & Hofer, H. W. (1988) J. Biol. Chem. 263, 952-957] and the mammalian enzyme.

Purification and characterization of gamma-glutamylcysteine synthetase from Ascaris suum.

We have purified and characterized the Ascaris suum gamma-glutamylcysteine synthetase, the rate-limiting step in the glutathione biosynthesis. The purified enzyme exhibited a specific activity of 18 U (mg protein)-1. Estimation of the molecular mass of the native enzyme by FPLC on Superdex S-200 revealed the presence of two enzyme activity peaks corresponding to molecular masses of 100 and 70 kDa. The higher-molecular-mass component could be dissociated by repeated gel filtration into the 70-kDa protein which is the enzymatically active subunit. The apparent Km values of the A. suum enzyme for L-aminobutyrate, L-cysteine and L-glutamate were 0.31, 0.41 and 0.94 mM, respectively. D,L-Buthionine-S,R-sulfoximine and cystamine showed time-dependent irreversible inhibitory effects on the A. suum enzyme activity with Ki values of 0.05 and 1.11 microM, respectively. The Ki values for the corresponding enzyme from rat kidney with D,L-buthionine-S,R-sulfoximine and cystamine were 7.19 and 22.2 microM, respectively. The time of half-inactivation of the enzyme at infinite concentration of D,L-buthionine-S,R-sulfoximine, tau 50, was determined to be 3.1 and 1.34 min, for the parasite and mammalian enzymes respectively. For cystamine, a tau 50 value of 3.32 min for the A. suum gamma-glutamylcysteine synthetase was determined, while a value of 2 min in case of rat kidney enzyme was found. The A. suum enzyme activity was competitively inhibited by glutathione with a Ki value of 0.11 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-base catalytic mechanism and pH dependence of fructose 2,6-bisphosphate activation of the Ascaris suum phosphofructokinase.

A form of phosphofructokinase (PFK) from Ascaris suum desensitized to hysteresis in the reaction time course and ATP allosteric inhibition has been used to study the activation by fructose 2,6-bisphosphate (F26P2) at varied pH in both reaction directions. In the direction of phosphorylation of F6P, V and V/KMgATP are constant over the pH range 6-9, while V/KF6P decreases at low pH, giving a pK value of 7.0, and at high pH, giving a pK of 8.9. V and V/KMgATP are insensitive to the presence of F26P2, but V/KF6P is increased by a constant amount in the presence of saturating F26P2 over the entire pH range studied. The concentration of F26P2 that gives half the change in V/KF6P, Kact, increases as the pH decreases, giving a pK of 7.4, reflecting an enzyme group that must be unprotonated for optimum binding of F26P2. In the direction of phosphorylation of MgADP, V and V/KMgADP are pH-independent, and both are insensitive to the presence of F26P2. V/KFBP decreases at high pH, giving a pK of about 7.3, and is increased by a constant amount in the presence of F26P2 over the entire pH range studied.(ABSTRACT TRUNCATED AT 250 WORDS)