Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.2.1.22 (cystathionine beta-synthase)
965 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Comparison of seven deduced amino acid sequences of cysteine synthase (O-acetyl-L-serine (thiol)-lyase, EC 4.2.99.8) from plants and bacteria disclosed the presence of 12 conserved Lys residues, which can be candidates for a functional binding site for pyridoxal phosphate cofactor. These 12 conserved Lys residues in a cDNA clone encoding spinach cysteine synthase A were replaced with Gly by oligonucleotide-directed in vitro mutagenesis. These Lys-->Gly mutated cDNAs were transferred into Escherichia coli NK3, a cysteine auxotroph lacking both cysteine synthase loci, cysK and cysM. One mutant replaced at Lys-49 could not complement the cysteine requirement of NK3, whereas other mutants and wild-type clone could. No enzymatic activity of cysteine synthase A was detected either in the cell-free extracts of E. coli NK3 transformed with the Lys-49 mutant. These results indicated that Lys-49 is a functional residue for the catalytic activity of cysteine synthase. This Lys residue is conserved in other evolutionarily related amino acid-metabolizing enzymes catalyzing reactions involving the beta-carbon of amino acids.
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PMID:Determination of a functional lysine residue of a plant cysteine synthase by site-directed mutagenesis, and the molecular evolutionary implications. 834 14

Homocysteine can be methylated to form methionine by the cobalamin- (Cbl) and folate-dependent enzyme, methionine synthase; serum levels of total homocysteine are elevated in greater than 95% of patients with either Cbl or folate deficiency. Homocysteine can also condense with serine to form cystathionine in a pyridoxal phosphate-dependent reaction catalyzed by cystathionine beta-synthase. Cystathionine is subsequently cleaved to cysteine and alpha-ketobutyrate by the pyridoxal phosphate-dependent enzyme gamma-cystathionase. To assess levels of cystathionine in Cbl and folate deficiency, we developed a new capillary gas chromatographic-mass spectrometric assay and measured cystathionine in the serum of normal subjects and patients with clinically confirmed deficiencies of these vitamins. The normal range for serum cystathionine was 65 to 301 nmol/L (median = 126 nmol/L) for 50 normal blood donors. In 30 patients with clinically confirmed Cbl deficiency, values for cystathionine ranged from 208 nmol/L to 2,920 nmol/L (median = 816 nmol/L) and 26 (87%) had levels above the normal range. In 20 patients with clinically confirmed folate deficiency, cystathionine concentrations ranged from 138 nmol/L to 4,150 nmol/L (median = 1,560 nmol/L) and 19 (95%) had values above the normal range. Five homozygotes for cystathionine beta-synthase deficiency had high values for serum-total homocysteine and low or low-normal values for serum cystathionine that ranged from 30 nmol/L to 114 nmol/L even though they were on treatment with pyridoxine and had partially responded. One patient with a defect in the synthesis of 5-CH3-tetrahydrofolate and five patients with defects in the synthesis of CH3-Cbl had high values for serum-total homocysteine and high values for cystathionine that ranged from 311 nmol/L to 1,500 nmol/L even though they were on treatment with folic acid and Cbl, respectively, and had partially responded. We conclude that levels of cystathionine are evaluated in the serum of most patients with Cbl and folate deficiency and that they are useful in the differential diagnosis of an elevated serum-total homocysteine level.
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PMID:Elevation of serum cystathionine levels in patients with cobalamin and folate deficiency. 850 76

The cysteine synthase gene (cysK) from Flavobacterium K3-15 was cloned and sequenced. The gene exhibits 30-50% identity to known cysteine synthases on both the DNA and the amino acid levels. The pyridoxal phosphate binding site of the enzyme is part of a conserved motif comprising seven amino acids (SIKDRIA). The lys31 residue of the flavobacterial enzyme is conserved in all known cysteine synthases. The cysK gene from Flavobacterium K3-15 was heterologously expressed and the gene product identified by immunoblotting and determination of the enzyme activity.
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PMID:Isolation of a gene encoding cysteine synthase from Flavobacterium K3-15. 886 84

The effect of concentrations of sulfur-containing amino acids, activities of cystathionine gamma-lyase and cystathionine beta-synthase, and level of vitamin B6 were examined following menthionine administration in normal rats and chronically uremic rats with 7/8 nephrectomy. In the uremic rats, the serum levels of methionine, cystathionine, cysteine and taurine increased in proportion to the amounts of methionine administered. The increase of taurine content in the serum and liver was particularly marked. Cystathionine beta-synthase activity in the liver increased with the administration, but the serum level of pyridoxal phosphate decreased markedly. The body weight gain of rats decreased with the administration, and the contents of urea and creatinine in serum increased. Thus, vitamin B6 deficiency in chronically uremic rats administered with large amounts of methionine may reduce growth, lower renal function and cause abnormal metabolism of sulfur-containing amino acids.
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PMID:Content of sulfur amino acids and vitamin B6 and related enzyme activities in rats with chronic renal failure fed a high methionine diet. 888 37

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.
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PMID:[Deregulation of homocysteine metabolism and consequences for the vascular system]. 923 30

Human cystathionine beta-synthase catalyzes the first step in the catabolic removal of the toxic metabolite, homocysteine. It is unique in being dependent on both pyridoxal phosphate (PLP) and heme for activity. The reaction involves condensation of serine and homocysteine to give cystathionine. Although the role of PLP can be rationalized in analogy with other PLP-dependent enzymes that catalyze beta-replacement reactions, the role of the heme is unknown. In this study, we have purified and characterized the recombinant human enzyme and have examined the effect of heme oxidation state on enzyme activity. We find that under reducing conditions, generated by addition of titanium citrate, the enzyme exhibits a 1.7-fold lower activity than under oxidizing conditions. Reoxidation of the ferrous enzyme with ferricyanide results in alleviation of inhibition. This redox-linked change in enzyme activity correlates with changes in heme oxidation state monitored by UV-visible spectroscopy. Dithiothreitol, which does not reduce the enzyme-bound heme, does not perturb enzyme activity. These studies provide the first evidence for redox-linked regulation of cystathionine beta-synthase which is heme-dependent.
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PMID:Evidence for heme-mediated redox regulation of human cystathionine beta-synthase activity. 973 78

Cystathionine beta-synthase is an unusual enzyme that requires the cofactors heme and pyridoxal phosphate (PLP) to catalyze the condensation of homocysteine and serine to generate cystathionine. This transsulfuration reaction represents one of two major cellular routes for detoxification of homocysteine, which is a risk factor for atherosclerosis. While the beta-replacement reaction catalyzed by this enzyme suggests a role for the pyridoxal phosphate, the role of the heme is uncertain. In this study we have examined the effect of changing one of the ligands to the heme on the activity of the enzyme. Binding of carbon monooxide results in the displacement of a thiolate ligand to the ferrous heme, and is accompanied by complete loss of cystathionine beta-synthase activity. Furthermore, inhibition by CO is competitive with respect to homocysteine, providing the first indication that the homocysteine binding site is in the proximity of heme. Binding of both CO and cyanide to ferrous cystathionine beta-synthase occurs in two distinct isotherms and indicates that the hemes are nonequivalent. We have employed fluorescence spectroscopy to characterize the bound PLP and its interaction with serine. PLP bound to cystathionine beta-synthase is weakly fluorescent and exists as a mixture of the protonated and unprotonated tautomers. Reaction with hydroxylamine releases the oxime and greatly enhances the associated fluorescence. Binding of serine is accompanied by a shift to the unprotonated tautomer of the external aldimine as well as the appearance of a new fluorescent species at approximately 400 nm that could be due to the aminoacrylate or to a gemdiamine intermediate. These data provide the first characterization of the PLP bound to cystathionine beta-synthase. Treatment of cystathionine beta-synthase with hydroxylamine releases two PLPs after 1 day and results in complete loss of activity. Incubation for an additional 3-4 days results in the release of two more PLPs. These data lead us to revise the PLP stoichiometry to 4 per tetramer, and to the conclusion that the heme and PLP sites in cystathionine beta-synthase are nonequivalent.
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PMID:Characterization of the heme and pyridoxal phosphate cofactors of human cystathionine beta-synthase reveals nonequivalent active sites. 1005 44

Cystathionine beta-synthase is a unique heme protein that catalyzes a pyridoxal phosphate (or PLP)-dependent beta-replacement reaction. The reaction involves the condensation of serine and homocysteine and constitutes one of the two major avenues for detoxification of homocysteine in mammals. The enzyme is allosterically regulated by S-adenosylmethionine (AdoMet). In this study, we have characterized the kinetic, spectroscopic, and ligand binding properties of a truncated catalytic core of cystathionine beta-synthase extending from residues 1 through 408 in which the C-terminal 143 residues have been deleted. This is similar to a natural variant of the protein that has been described in a homocystinuric patient in which the predicted peptide is 419 amino acids in length. Truncation leads to the formation of a dimeric enzyme in contrast to the tetrameric organization of the native enzyme. Some of the kinetic properties of the truncated enzyme are different from the full-length form, most notably, significantly higher K(m)s for the two substrates, and loss of activation by AdoMet. This is paralleled by the absence of AdoMet binding to the truncated form, whereas four AdoMet molecules bind cooperatively to the full-length tetrameric enzyme with a K(d) of 7. 4 microM. Steady-state kinetic analysis indicates that the order of substrate addition is important. Thus, preincubation of the enzyme with homocysteine leads to a 2-fold increase in V(max) relative to preincubation of the enzyme with serine. Since the intracellular concentration of serine is significantly greater than that of homocysteine, the physiological significance of this phenomenon needs to be considered. Based on ligand binding studies and homology searches with protein sequences in the database, we assign residues 68-209 as being important for PLP binding, residues 241-341 for heme binding, and residues 421-469 for AdoMet binding.
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PMID:Assignment of enzymatic functions to specific regions of the PLP-dependent heme protein cystathionine beta-synthase. 1052 87

The most common cause of severely elevated homocysteine or homocystinuria is inherited disorders in cystathionine beta-synthase. The latter enzyme is a unique hemeprotein that catalyzes pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine, thus committing homocysteine to catabolism. A point mutation, V168M, has been described in a homocystinuric cell line and is associated with a B(6)-responsive phenotype. In this study, we have examined the kinetic properties of this mutant and demonstrate that the mutation affects the PLP but not the heme content. The approximately 13-fold diminution in activity because of the mutation corresponds to an approximately 7-fold decrease in the level of bound PLP. This may be explained by half of the sites activity associated with cystathionine beta-synthase. The addition of PLP results in partial but not full restoration of activity to wild type levels. Elimination of the C-terminal quarter of the mutant protein results in alleviation of the catalytic penalty imposed by the V168M mutation. The resulting truncated protein is very similar to the corresponding truncated enzyme with wild type sequence and is now able to bind the full complement of both heme and PLP cofactors. These results indicate that the V168M mutation per se does not affect binding of PLP directly and that interactions between the regulatory C terminus and the catalytic N terminus are important in modulating the cofactor content and therefore the activity of the full-length enzyme. These studies provide the first biochemical explanation for the B(6)-responsive phenotype associated with a cystathionine beta-synthase-impaired homocystinuric genotype.
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PMID:Deletion of the regulatory domain in the pyridoxal phosphate-dependent heme protein cystathionine beta-synthase alleviates the defect observed in a catalytic site mutant. 1053 22

Various vitamin B6 enzymes play important roles in mammalian and microbial metabolism of selenium amino acids. Selenocysteine is synthesized from selenohomocysteine by catalysis of cystathionine beta-synthase and cystathionine gamma-lyase, which both require pyridoxal phosphate. Selenocysteine beta-lyase, a new B6-enzyme, exclusively catalyzes beta-elimination of selenocysteine, and occurs in mammalian systems and bacteria. Methionine gamma-lyase, cysteine desulfurase, cysteine sulfinate desulfinase, and D-selenocystine alpha,beta-lyase, which are B6-enzymes, act on cysteine, cysteine sulfinate, D-cystine, and their derivatives, and their selenium counterparts indiscriminately. Their reaction mechanisms are comparatively described.
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PMID:Vitamin B6 enzymes participating in selenium amino acid metabolism. 1060 91


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