Deregulation of homocysteine metabolism and consequences for the vascular system

The link between vascular disease and elevated homocysteine levels has been recognized for more than 30 years, and association with moderately elevated levels has been suspected for 20 years. Homocysteine is a sulfhydryl-containing amino acid that is formed by the demethylation of methionine. It is normally catalysed to cystathionine by cystathionine beta-synthase a pyridoxal phosphate-dependent enzyme. Homocysteine is also remethylated to methionine by methionine synthase, a vitamin B12 dependent enzyme and by methylenetetrahydrofolate reductase. Environmental factors such as folate, or vitamin B12, or vitamin B6 deficiencies and genetic defects such as cystathionine beta-synthase or abnormality of methylene-tetrahydrofolate reductase or some vitamin B12 metabolism defects may contribute to increasing plasma homocysteine levels. Normal fasting levels of homocysteine lie within the range 6-16 mumol/l. Apart from differences in assay methods, age, sex and nutritional status may affect the plasma levels. Though it is now well known that homocysteine is an independent risk factor for premature vascular disease, the pathogenesis of homocysteine-induced vascular damage is, for the most part, unknown. It may be multifactorial, including direct homocysteine damage to the endothelium, an enhanced low-density lipoprotein peroxidation, an increase of platelet thromboxane A2, or a decrease of protein C activation.     

Disordered methionine/homocysteine metabolism in premature vascular disease. Its occurrence, cofactor therapy, and enzymology

Mild homocysteinemia occurs surprisingly often in patients with premature vascular disease. We studied the possible enzymatic sources of this mild hyperhomocysteinemia and the control of homocysteine levels in plasma by treatment of patients with the cofactors and cosubstrates of homocysteine catabolism. We assessed homocysteine metabolism in 131 patients who had premature disease in their coronary, peripheral, or cerebrovascular circulation by using a standard oral methionine-load test. Impaired homocysteine metabolism occurred in 28 patients. We assayed levels of the primary enzymes of homocysteine catabolism in cultured skin fibroblast extracts from 15 of these 28 patients. The patients' cystathionine beta-synthase levels (3.68 +/- 2.52 nmol/h per milligram of cell protein, mean +/- SD) were markedly depressed compared with those from 31 healthy adult control subjects (7.61 +/- 4.49, P < .001). The patients' levels of 5-methyltetrahydrofolate: homocysteine methyltransferase were normal. While betaine: homocysteine methyltransferase was not expressed in skin fibroblasts, 24-hour urinary betaine and N,N-dimethylglycine measurements were consistent with normal or enhanced remethylation of homocysteine by betaine: homocysteine methyltransferase in the 13 patients tested. When treated daily with choline and betaine, pyridoxine, or folic acid, there was a normalization of the postmethionine plasma homocysteine level in 16 of 19 patients. Our results indicate that mild homocysteinemia in premature vascular disease may be caused by either a folate deficiency or deficiencies in cystathionine beta-synthase activity. It does not necessarily involve deficiencies of either 5-methyltetrahydrofolate:homocysteine methyltransferase or betaine:homocysteine methyltransferase. Effective treatment regimens are also defined.     

Effects of streptozotocin-induced diabetes and of insulin treatment on homocysteine metabolism in the rat

An elevation in the concentration of total plasma homocysteine is known to be an independent risk factor for the development of vascular disease. Alterations in homocysteine metabolism have also been observed clinically in diabetic patients. Patients with either type 1 or type 2 diabetes who have signs of renal dysfunction tend to exhibit elevated total plasma homocysteine levels, whereas type 1 diabetic patients who have no clinical signs of renal dysfunction have lower than normal plasma homocysteine levels. The purpose of this study was to investigate homocysteine metabolism in a type 1 diabetic animal model and to examine whether insulin plays a role in its regulation. Diabetes was induced by intravenous administration of 100 mg/kg streptozotocin to Sprague-Dawley rats. We observed a 30% reduction in plasma homocysteine in the untreated diabetic rat. This decrease in homocysteine was prevented when diabetic rats received insulin. Transsulfuration and remethylation enzymes were measured in both the liver and the kidney. We observed an increase in the activities of the hepatic transsulfuration enzymes (cystathionine beta-synthase and cystathionine gamma-lyase) in the untreated diabetic rat. Insulin treatment normalized the activities of these enzymes. The renal activities of these enzymes were unchanged. These results suggest that insulin is involved in the regulation of plasma homocysteine concentrations by affecting the hepatic transsulfuration pathway, which is involved in the catabolism of homocysteine.     

Disorders of homocysteine metabolism

This review of recent advances covers (1) the metabolism of methionine and its regulation, emphasizing interactions with the three important vitamins folate, cobalamin and pyridoxine; (2) present knowledge of enzymological and moleculargenetic aspects of homozygous deficiencies of the three enzymes which cause elevated homocyst(e)ine; (3) recent clinical findings, post-methionine loading results related to enzyme and mutation studies in obligate heterozygotes for cystathionine beta-synthase deficiency; (4) important new evidence for disturbed homocysteine metabolism in neural tube defects, particularly based on studies of the thermolabile methylene-tetrahydrofolate reductase mutation which is also of importance in vascular disease; (5) the suitability and limitations of animal models that have so far been described.     

Homocysteine metabolism in endothelial cells of a patient homozygous for cystathionine beta-synthase (CS) deficiency

Homocystinuria due to cystathionine beta-synthase (CS) deficiency is the most common inborn error of methionine metabolism. Patients with CS-deficiency have an extremely high risk of vascular disease. The underlying mechanism is still unsolved. Dysfunction of endothelial cells could be the trigger in the formation of atherosclerosis and thrombosis. Therefore, differences in cell function were studied between normal and CS-deficient human umbilical endothelial cells (HUVECs). Total homocysteine (tHcy) concentrations in culture media as a measure of homocysteine export increased in all cell lines, including the cell line with CS-deficiency, with constant amounts of approximately 2.5 microM every 24 h. von Willebrand factor (vWF), tissue plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) in culture media were used as markers of endothelial function and increased also with progression of culture time. The effects of additions of folate, vitamin B6 and methionine to the culture medium were studied. The homocysteine export and the markers of endothelial function did not differ between the control and the CS-deficient HUVECs under various test conditions. These data show that CS-deficient endothelial cells have normal homocysteine export and normal endothelial cell function. In CS-deficient patients the very high blood levels of homocysteine, probably due to deficient CS function in liver and kidney, seems to be the hazardous factor to endothelial cells, thus promoting atherosclerosis and thrombosis in CS-deficient patients.     

Metabolism of homocysteine thiolactone in human cell cultures. Possible mechanism for pathological consequences of elevated homocysteine levels

Editing of the non-protein amino acid homocysteine, a frequent type of error-correcting process in amino acid selection for protein synthesis by an aminoacyl-tRNA synthetase, results in formation of a cyclic thioester, homocysteine thiolactone. Here it is shown that human cells in which homocysteine metabolism is deregulated by a mutation in the cystathionine beta-synthase gene and/or by an antifolate drug, aminopterin (which prevents remethylation of homocysteine to methionine by methionine synthase), produce more homocysteine thiolactone, in addition to homocysteine, than unaffected cells. The thiolactone is incorporated into cellular and extracellular proteins, in addition to being secreted and hydrolyzed to homocysteine. Experiments with model proteins and amino acids suggest that the mechanism of incorporation involves acylation of side chain amino groups of lysine residues by the activated carboxyl group of the thiolactone. The metabolic conversion of homocysteine to homocysteine thiolactone and the reactivity of the thiolactone toward proteins may explain pathological consequences of elevated levels of homocysteine such as observed in vascular disease.     

Functionalized de novo designed proteins: mechanism of proton coupling to oxidation/reduction in heme protein maquettes

Proton exchange with aqueous media coupled to heme oxidation/reduction is commonly seen but not understood in natural cytochromes. Our synthetic tetrahelix bundle heme protein maquettes successfully reproduce natural proton coupling to heme oxidation/reduction. Potentiometry reveals major pK shifts from 4.2 to 7.0 and from 9.4 to 10.3 in the maquette-associated acid/base group(s) upon heme reduction. Consequently, a 210 mV decrease in the heme redox potential is observed between the two extremes of pH. Potentiometry with resonance Raman and FTIR spectroscopy performed over a wide pH range strongly implicates glutamate side chains as the source of proton coupling below pH 8.0, whereas lysine side chains are suggested above pH 8.0. Remarkably, the pK values of several glutamates in the maquette are elevated from their solution value (4.4) to values as high as 7.0. It is suggested that these glutamates are recruited into the interior of the bundle as part of a structural rearrangement that occurs upon heme binding. Glutamate to glutamine variants of the prototype protein demonstrate that removal of the glutamate closest to the heme diminishes but does not abolish proton exchange. It is necessary to remove additional glutamates before pH-independent heme oxidation/reduction profiles are achieved. The mechanism of redox-linked proton coupling appears to be rooted in distributed partial charge compensation, the magnitude of which is governed by the dielectric distance between the ferric heme and acid/base side chains. A similar mechanism is likely to exist in native redox proteins which undergo charge change upon cofactor oxidation/reduction.     

Oxygen and one reducing equivalent are both required for the conversion of alpha-hydroxyhemin to verdoheme in heme oxygenase

Heme oxygenase is a central enzyme of heme degradation and associated carbon monoxide biosynthesis. We have prepared the alpha-hydroxyheme-heme oxygenase complex, which is the first intermediate in the catalytic reaction. The active site structure of the complex was examined by optical absorption, EPR, and resonance Raman spectroscopies. In the ferric form of the enzyme complex, the heme iron is five coordinate high spin and the alpha-hydroxyheme group in the complex assumes a structure of an oxophlorin where the alpha-meso hydroxy group is deprotonated. In the ferrous form, the alpha-hydroxy group is protonated and consequently the prosthetic group assumes a porphyrin structure. The alpha-hydroxyheme group undergoes a redox-linked conversion between a keto and an enol form. The ferric alpha-hydroxyheme reacts with molecular oxygen to form a radical species. Reaction of the radical species with a reducing equivalent yields the verdoheme-heme oxygenase complex. Reaction of the ferrous alpha-hydroxyheme-heme oxygenase complex with oxygen also yields the verdoheme-enzyme complex. We conclude that the catalytic conversion of ferric alpha-hydroxyheme to verdoheme by heme oxygenase requires molecular oxygen and one reducing equivalent.     

Intramolecular electron transfer in yeast flavocytochrome b2 upon one-electron photooxidation of the fully reduced enzyme: evidence for redox state control of heme-flavin communication

Flavocytochrome b2, which has been fully reduced using L-lactate, can be rapidly oxidized by 1 equiv using the laser-generated triplet state of 5-deazariboflavin. Parallel photoinduced oxidation occurs at the reduced heme and at the fully reduced FMN (FMNH2) prosthetic groups of different enzyme monomers, producing the anion semiquinone of FMN and a ferric heme. Following the initial oxidation reaction, rapid intramolecular reduction of the ferric heme occurs with concomitant oxidation of FMNH2, generating the neutral FMN semiquinone. The observed rate constant for this intramolecular electron transfer is 2200 s-1, which is 1 order of magnitude larger than the turnover number under these conditions. A slower reduction of the heme prosthetic group also occurs with an observed rate constant of approximately 10 s-1, perhaps due to intersubunit electron transfer from reduced FMN to heme. The rapid intramolecular electron transfer between the FMNH2 and ferric heme is eliminated upon addition of excess pyruvate (Ki = 3.8 mM). This latter result indicates that pyruvate inhibition of catalytic turnover apparently can occur at the FMNH2-->heme electron transfer step. These results markedly differ from those previously obtained (Walker, M. C., & Tollin, G. (1991) Biochemistry 30, 5546-5555) and confirmed here for electron transfer within the one-electron reduced enzyme and for the effect of pyruvate binding, suggesting that intramolecular communication between the heme and flavin prosthetic groups can be controlled by the redox state of the enzyme and by ligand binding to the active site.     

Cloning and expression of cDNA for soluble form of rat heme oxygenase-1

Heme oxygenase catalyzes the oxidation of heme to biliverdin and carbon monoxide. The gene encoding the truncated soluble rat heme oxygenase-1 (Metl-Pro267) was cloned. The enzyme protein was expressed in E. coli JM109 and purified to homogeneity. The molecular weight of the recombinant enzyme was 30 kDa as assessed by SDS-polyacrylamide gel electrophoresis. From a 3-L culture, about 90 mg of the purified enzyme was routinely obtained. The dependency of the heme oxygenase reaction catalyzed by the soluble enzyme on the NADPH-cytochrome P-450 reductase concentrations and the effect of catalase on the reaction were examined to compare with the purified membrane-bound form of heme oxygenase-1 (Yoshida and Kikuchi, 1978b). The activity of the soluble enzyme was inhibited at high concentrations of NADPH-cytochrome P-450 reductase and the inhibition was not alleviated by addition of catalase unlike the membrane-bound form. The ferric iron of the heme-heme oxygenase complex was in a typical high spin state at acidic to neutral pH (pH 6.5-7.0) but conversion to low spin state was observed at basic pH (pH 9-10). The heme bound to heme oxygenase was converted to biliverdin at a stoichiometric ratio of unity in the presence of NADPH-cytochrome P-450 reductase system. During the heme degradation of the heme-heme oxygenase complex under atmospheric oxygen, several intermediates, that is, oxygenated heme and verdoheme, were spectrally discriminated.     

Kinase activity of oxygen sensor FixL depends on the spin state of its heme iron

FixL is a ferrous heme protein whose kinase activity is inhibited by oxygen. Here we show that met-FixL, which is the ferric unliganded form, has the same activity as deoxy-FixL, the ferrous unliganded form, indicating that activity does not depend on the oxidation state of the heme iron. The ferric derivative fluoro-FixL is fully active, indicating that the presence of a heme ligand is not sufficient to cause kinase inhibition. An inverse relation between the rate of autophosphorylation of ferric FixL and the fractional saturation with cyanide shows that the cyanomet form has zero activity. All our active derivatives were high-spin, while our inactive derivatives were low-spin. In mixtures of high- and low-spin FixL, resulting from partial saturation with low-spin ligands, the activity was that which would be expected for the concentration of the high-spin component alone. Therefore the spin state of the heme iron rather than the oxidation state or presence of ligands must be the factor that controls FixL's kinase activity. On transition from low to high spin, the heme iron moves out of the porphyrin plane by 0.4 A. We propose that, as in hemoglobin, this motion triggers a long-range conformational change which in FixL is responsible for a switch to an active form.     

Trypsin cleavage of human cystathionine beta-synthase into an evolutionarily conserved active core: structural and functional consequences

Cystathionine beta-synthase (CBS) catalyzes the condensation of homocysteine and serine to cystathionine-an irreversible step in the eukaryotic transsulfuration pathway. The native enzyme is a homotetramer or multimer of 63-kDa (551 amino acids) subunits and is activated by S-adenosyl-l-methionine (AdoMet) or by partial cleavage with trypsin. Amino-terminal analysis of the early products of trypsinolysis demonstrated that the first cleavages occur at Lys 30, 36, and 39. The enzyme still retains the subunit organization as a tetramer or multimer composed of 58-kDa subunits. Analysis by electrospray ionization mass spectrometry showed that further trypsin treatment cleaves CBS in its COOH-terminal region at Arg 413 to yield 45-kDa subunits. This 45-kDa active core is the portion of CBS most conserved with the evolutionarily related enzymes isolated from plants, yeast, and bacteria. The active core of CBS forms a dimer of approximately 85 kDa. The dimer is about twice as active as the tetramer. It binds both pyridoxal 5'-phosphate and heme cofactors but is no longer activated by AdoMet. Further analysis suggests that the dissociation of CBS to dimers causes a decrease in enzyme thermostability and a threefold increase in affinity toward the sulfhydryl-containing substrate-homocysteine. We found that the COOH-terminal region, residues 414-551, is essential for maintaining the tetrameric structure and AdoMet activation of the enzyme. The inability of the active core to form multimeric aggregates has facilitated its crystallization and X-ray diffraction studies.     

Crystal structure of Escherichia coli cystathionine gamma-synthase at 1.5 A resolution

The transsulfuration enzyme cystathionine gamma-synthase (CGS) catalyses the pyridoxal 5'-phosphate (PLP)-dependent gamma-replacement of O-succinyl-L-homoserine and L-cysteine, yielding L-cystathionine. The crystal structure of the Escherichia coli enzyme has been solved by molecular replacement with the known structure of cystathionine beta-lyase (CBL), and refined at 1.5 A resolution to a crystallographic R-factor of 20.0%. The enzyme crystallizes as an alpha4 tetramer with the subunits related by non-crystallographic 222 symmetry. The spatial fold of the subunits, with three functionally distinct domains and their quaternary arrangement, is similar to that of CBL. Previously proposed reaction mechanisms for CGS can be checked against the structural model, allowing interpretation of the catalytic and substrate-binding functions of individual active site residues. Enzyme-substrate models pinpoint specific residues responsible for the substrate specificity, in agreement with structural comparisons with CBL. Both steric and electrostatic designs of the active site seem to achieve proper substrate selection and productive orientation. Amino acid sequence and structural alignments of CGS and CBL suggest that differences in the substrate-binding characteristics are responsible for the different reaction chemistries. Because CGS catalyses the only known PLP-dependent replacement reaction at Cgamma of certain amino acids, the results will help in our understanding of the chemical versatility of PLP.     

The primitive protozoon Trichomonas vaginalis contains two methionine gamma-lyase genes that encode members of the gamma-family of pyridoxal 5'-phosphate-dependent enzymes

Methionine gamma-lyase, the enzyme that catalyzes the breakdown of methionine by an alpha,gamma-elimination reaction and is a member of the gamma-family of pyridoxal 5'-phosphate-dependent enzymes, is present in high activity in the primitive protozoan parasite Trichomonas vaginalis but is absent from mammals. Two genes, mgl1 and mgl2, encoding methionine gamma-lyase, have now been isolated from T. vaginalis. They are both single copy, encode predicted proteins (MGL1 and MGL2) of 43 kDa, have 69% sequence identity with each other, and show a high degree of sequence identity to methionine gamma-lyase from Pseudomonas putida (44%) and other related pyridoxal 5'-phosphate-dependent enzymes such as human cystathionine gamma-lyase (42%) and Escherichia coli cystathionine beta-lyase (30%). mgl1 and mgl2 have been expressed in E. coli as a fusion with a six-histidine tag and the recombinant proteins (rMGL1 and rMGL2) purified by metal-chelate affinity chromatography. rMGL1 and rMGL2 were found to have high activity toward methionine (10.4 and 0.67 mumol/min/mg of protein, respectively), homocysteine (370 and 128 mumol/min/mg of protein), cysteine (6.02 and 1.06 mumol/min/mg of protein), and O-acetylserine (3.74 and 1.51 mumol/min/mg of protein), but to be inactive toward cystathionine. Site-directed mutagenesis of an active site cysteine (C113G for MGL1 and C116G for MGL2) reduced the activity of the recombinant enzymes toward both methionine and homocysteine by approximately 80% (rMGL1) and 90% (rMGL2). In contrast, the activity of mutated rMGL2 toward cysteine and O-acetylserine was increased (to 214 and 142%, respectively), whereas that of mutated rMGL1 was reduced to 39 and 49%, respectively. These findings demonstrate the importance of this cysteine residue in the alpha,beta-elimination and alpha, gamma-elimination reactions catalyzed by trichomonad methionine gamma-lyase.     

Comparative functioning of dihydro- and tetrahydropterins in supporting electron transfer, catalysis, and subunit dimerization in inducible nitric oxide synthase

The nitric oxide synthases (NOS) are the only heme-containing enzymes that require tetrahydrobiopterin (BH4) as a cofactor. Previous studies indicate that only the fully reduced (i.e., tetrahydro) form of BH4 can support NO synthesis. Here, we characterize pterin-free inducible NOS (iNOS) and iNOS reconstituted with eight different tetrahydro- or dihydropterins to elucidate how changes in pterin side-chain structure and ring oxidation state regulate iNOS. Seven different enzyme properties that are important for catalysis and are thought to involve pterin were studied. Only two properties were found to depend on pterin oxidation state (i.e., they required fully reduced tetrahydropterins) and were independent of side chain structure: NO synthesis and the ability to increase heme-dependent NADPH oxidation in response to substrates. In contrast, five properties were exclusively dependent on pterin side-chain structure or stereochemistry and were independent of pterin oxidation state: pterin binding affinity, and its ability to shift the heme iron to its high-spin state, stabilize the ferrous heme iron coordination structure, support heme iron reduction, and promote iNOS subunit assembly into a dimer. These results clarify how structural versus redox properties of the pterin impact on its multifaceted role in iNOS function. In addition, the data reveal that during NO synthesis all pterin-dependent steps up to and including heme iron reduction can take place independent of the pterin ring oxidation state, indicating that the requirement for fully reduced pterin occurs at a point in catalysis beyond heme iron reduction.     

L-thiocitrulline. A stereospecific, heme-binding inhibitor of nitric-oxide synthases

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (NO). The enzyme is inhibited by a variety of N omega-monosubstituted L-arginine analogs, and some of these compounds are useful in reversing pathologies associated with the overproduction of NO (e.g. the hypotension of septic shock). We report here that L-thiocitrulline (gamma-thioureido-L-norvaline) is a potent, stereospecific inhibitor of the constitutive brain and endothelial isoforms of NOS as well as the isoform induced in vascular smooth muscle cells by lipopolysaccharide and interferon-gamma. Steady state kinetic studies show L-thiocitrulline inhibition is competitive with L-arginine (Ki approximately 4-20% of KArgm), indicating that initial binding is as a substrate/product analog. In contrast to L-arginine and N omega-methyl-L-arginine, the prototypic NOS inhibitor, L-thiocitrulline binding elicits a "Type II" difference spectrum, indicating a high spin to low spin transition of the iron in the heme cofactor. This finding suggests that L-thiocitrulline is contributing the sixth ligand to heme iron, probably through the thioureido sulfur. Such interaction with heme iron neither stimulates nor inhibits the direct flavin-mediated cytochrome c reduction activity of the enzyme, but it does inhibit heme-dependent superoxide formation. In vivo, L-thiocitrulline is a potent pressor agent in both normal and endotoxemic rats, the latter finding suggesting utility in treating the hypotension of septic shock.     

Altering substrate specificity at the heme edge of cytochrome c peroxidase

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibit unique specificities toward oxidation of small substrates. Ala-147 in CCP is located near the delta-meso edge of the heme and along the solvent access channel through which H2O2 is thought to approach the active site. This residue was replaced with Met and Tyr to investigate the hypothesis that small molecule substrates are oxidized at the exposed delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta-meso heme position and might therefore modify small molecule access to the oxidized heme cofactor. Steady-state kinetic measurements show that cytochrome c oxidation is enhanced 3-fold for A147Y relative to wild type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of phenols by A147Y is reduced to less than 20% relative to the wild-type enzyme, while Vmax/e for oxidation of other small molecules is less affected by either mutation. However, the "specificity" of aniline oxidation by A147M, i.e., (Vmax/e)/Km, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been introduced by the mutation. Stopped-flow kinetic data show that the restricted heme access in A147Y or A147M slows the reaction between the enzyme and H202, but not to an extent that it becomes rate limiting for the oxidation of the substrates examined. The rate constant for compound ES formation with A147Y is 2.5 times slower than wild-type CCP. These observations strongly support the suggestion that small molecule oxidations occur at sites on the enzyme distinct from those utilized by cytochrome c and that the specificity of small molecule oxidation can be significantly modulated by manipulating access to the heme edge. The results help to define the role of alternative electron transfer pathways in cytochrome c peroxidase and may have useful applications in improving the specificity of peroxidase with engineered function.     

Parasite sulphur amino acid metabolism

This paper reviews current knowledge regarding the metabolism of the sulphur-containing amino acids methionine and cysteine in parasitic protozoa and helminths. Particular emphasis is placed on the unusual aspects of parasite biochemistry which may present targets for rational design of antiparasite drugs. In general, the basic pathways of sulphur amino acid metabolism in most parasites resemble those of their mammalian hosts, since the enzymes involved in (a) the methionine cycle and S-adenosylmethionine metabolism, (b) the trans-sulphuration sequence, (c) the transminative catabolism of methionine, (d) the oxidative catabolism of cysteine and (e) glutathione synthesis have been demonstrated variously in several helminth and protozoan species. Despite these common pathways, there also exist numerous differences between parasite and mammalian metabolism. Some of these differences are relatively subtle. For example, the biochemical properties (and primary amino acid structures) of certain parasite methionine cycle enzymes and S-adenosylmethionine decarboxylases differ from those of the corresponding mammalian enzymes, and nematodes and trichomonads possess a novel, non-mammalian form of the trans-sulphuration enzyme cystathionine beta-synthase. The most profound differences between parasite and mammalian biochemistry relate to a number of unusual enzymes and thiol metabolites found in parasitic protozoa. In certain protozoa the pathway for methionine recycling from 5'-methylthioadenosine differs markedly from the mammalian route, and involves 2 exclusively microbial enzymes. Trypanosomatid protozoa contain the non-mammalian antioxidant thiol compounds ovothiol A and trypanothione, together with unique trypanothione-linked enzymes. Specific anaerobic protozoa possess another exclusively microbial enzyme, methionine gamma-lyase, which catabolises methionine (and homocysteine); the physiological significance of these non-mammalian activities is not fully understood. These unusual features offer opportunities for chemotherapeutic exploitation, and in some cases represent metabolic similarities with bacteria. Additionally, some anaerobic protozoa contain unidentified thiols and this implies the presence of further unusual enzymes/pathways in these organisms. So far, no truly unique targets for chemotherapy have been found in helminth sulphur amino acid metabolism, and to some degree this reflects the relative lack of detailed study in the area.     

Oxidation of catalase by singlet oxygen

Different bands of catalase activity in zymograms (Cat-1a-Cat-1e) appear during Neurospora crassa development and under stress conditions. Here we demonstrate that singlet oxygen modifies Cat-1a, giving rise to a sequential shift in electrophoretic mobility, similar to the one observed in vivo. Purified Cat-1a was modified with singlet oxygen generated from a photosensitization reaction; even when the reaction was separated from the enzyme by an air barrier, a condition in which only singlet oxygen can reach the enzyme by diffusion. Modification of Cat-1a was hindered when reducing agents or singlet oxygen scavengers were present in the photosensitization reaction. The sequential modification of the four monomers gave rise to five active catalase conformers with more acidic isoelectric points. The pI of purified Cat-1a-Cat-1e decreased progressively, and a similar shift in pI was observed as Cat-1a was modified by singlet oxygen. No further change was detected once Cat-1e was reached. Catalase modification was traced to a three-step reaction of the heme. The heme of Cat-1a gave rise to three additional heme peaks in a high performance liquid chromatography when modified to Cat-1c. Full oxidation to Cat-1e shifted all peaks into a single one. Absorbance spectra were consistent with an increase in asymmetry as heme was modified. Bacterial, fungal, plant, and animal catalases were all susceptible to modification by singlet oxygen, indicating that this is a general feature of the enzyme that could explain in part the variety of catalases seen in several organisms and the modifications observed in some catalases. Modification of catalases during development and under stress could indicate in vivo generation of singlet oxygen.