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)

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.
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PMID:Trypsin cleavage of human cystathionine beta-synthase into an evolutionarily conserved active core: structural and functional consequences. 967 31

The thiol amino acid homocysteine (HC) accumulates in homocystinuria and homocyst(e)inemia, and is associated with a wide variety of clinical manifestations. To determine whether HC influences the cell's program of gene expression, vascular endothelial cells were treated with HC for 6-42 h and analyzed by differential display. We found a 3-7-fold, time-dependent induction of a 220-base pair fragment, which demonstrated complete sequence identity with elongation factor-1delta (EF-1delta), a member of the multimeric complex regulating mRNA translation. Fibroblasts from cystathionine beta-synthase -/- individuals also showed up to 3.0-fold increased levels of mRNA for EF-1alpha, -beta, and -delta when compared with normal cells, and treatment of normal cells with the HC precursor, methionine, induced a 1.5-2.0-fold increase in EF-1alpha, -beta, and -delta mRNA. This induction was completely inhibited by cycloheximide and reflected a doubling in the rate of gene transcription in nuclear run-on analyses. In HC-treated endothelial cells, pulse-chase studies revealed a doubling in the rate of synthesis of the thiol-containing protein, annexin II, but no change in synthesis of the cysteineless protein, plasminogen activator inhibitor-1. Thus, HC induces expression of a family of acute translational response genes through a protein synthesis-dependent transcriptional mechanism. This process may mediate accelerated synthesis of free thiol-containing proteins in response to HC-induced oxidative stress.
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PMID:Induction of acute translational response genes by homocysteine. Elongation factors-1alpha, -beta, and -delta. 967 19

In order to determine if cystathionine beta-synthase (CBS) could separate groups of patients with various vascular disease, CBS activity was studied in cultured human skin fibroblasts from 30 subjects being either controls, atherosclerotic patients or patients having suffered a deep venous thrombosis. We found a tendency to a negative correlation between age and CBS activity in the control group only (r = -0.58, P = 0.08), with a tendency to lower CBS activities in the young patients with atherosclerotic (4.9) or venous disease (5.3) compared to the young control group (10.2). This could implicate higher levels of p-homocysteine with increased age as well as in young patients with atherosclerotic or thrombotic disease causing vascular damage. The results are important for the further discussion of the role of homocysteine as a risk factor for developing atherosclerotic and thrombogenic vascular disease and for finding a suitable screening method as prevention is by vitamin supplement only.
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PMID:Age and cystathionine beta-synthase activity in cultured fibroblasts from patients with arterial and venous vascular disease. 971 28

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 [CBS; l-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration and is the enzyme deficient in classical homocystinuria. In this report, we describe the molecular cloning and the complete nucleotide sequence of the human CBS gene. We report a total of 28,046 nucleotides of sequence, which, in addition to the CBS gene, contains approximately 5 kb of the 5' flanking region. The human CBS gene contains 23 exons ranging from 42 to 209 bp. The 5' UTR is formed by 1 of 5 alternatively used exons and 1 invariably present exon, while the 3' UTR is encoded by exons 16 and 17. We also describe the identification of two alternatively used promoter regions that are GC rich (approximately 80%) and contain numerous putative binding sites for Sp1, Ap1, Ap2, and c-myb, but lack the classical TATA box. The CBS locus contains an unusually high number of Alu repeats, which may predispose this gene to deleterious rearrangements. Additionally, we report on a number of DNA sequence repeats that are polymorphic in North American and European Caucasians.
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PMID:The human cystathionine beta-synthase (CBS) gene: complete sequence, alternative splicing, and polymorphisms. 979 Jul 50

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.
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PMID:Effects of streptozotocin-induced diabetes and of insulin treatment on homocysteine metabolism in the rat. 983 32

Recent developments in ultrasound technology enable the noninvasive measurement of structural and functional vessel wall changes. Until now, the effect of homocysteine on the arterial wall has remained unclear: reports on intima-media thickness (IMT) yield conflicting results, whereas data on vessel wall stiffness are lacking. Because several cardiovascular risk factors result in an increased IMT or stiffness, different groups at risk for atherosclerotic disease, with special emphasis on hyperhomocysteinemia, were studied. Nineteen patients homozygous and 14 subjects heterozygous for cystathionine beta-synthase (CBS) deficiency, 21 patients with familial hypercholesterolemia (FH), 15 patients with essential hypertension, 20 smokers, and 28 control subjects were studied. The IMT values (both right and left) of the common carotid artery (CCA), bulb (BUL), internal carotid artery (ICA), and common femoral artery (CFA) were measured in millimeters by high-resolution ultrasound (Biosound). The distensibility (DC, in 10(-3). kPa-1) and compliance (CC in mm2. kPa-1) coefficients of the CCA (right and left) and CFA (right) were determined by a wall track system (Pie Medical). The mean IMT of the posterior wall in the CCA was 0.70+/-0.09 mm in healthy controls. For patients with vascular disease, FH, and hypertension and in smokers, the mean CCA IMT was larger, whereas no major differences in IMT were observed in patients either homozygous or heterozygous for CBS deficiency. The DC and CC in the right CCA were 23.5+/-6.9 (10(-3). kPa-1) and 0.9+/-0.3 (mm2. kPa-1) in healthy subjects, slightly lower in patients homozygous for CBS deficiency, and clearly lower in patients with vascular disease, FH, and hypertension. No positive correlation was found between plasma homocysteine level and either IMT, CC, or DC. Because smoking was a confounder in each risk group, a stepwise regression analysis was carried out to assess the contribution of each risk factor on IMT and arterial wall stiffness. Age explained most of the variation in IMT of the CCA (coefficient of determination R2 of 0.34), whereas R2 values for serum low density lipoprotein cholesterol, smoking (pack-years), and systolic blood pressure were 0.08, 0.07, and 0.06, respectively. Homocysteine did not contribute to variation in IMT in both the CCA and CFA. Age and smoking contributed to the variation in IMT in the CFA. The variation in DC and CC in the right CCA and right CFA could in part be explained by age, low density lipoprotein cholesterol, and blood pressure. Plasma homocysteine concentration explained only a small proportion of the variation in DC in the CCA (R2=0.02) and in CC in the CFA (R2=0.04). In this study, no relationship was found between homocysteine level and the thickness of the arterial wall, with only a marginal influence on stiffness.
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PMID:Carotid and femoral artery wall thickness and stiffness in patients at risk for cardiovascular disease, with special emphasis on hyperhomocysteinemia. 984 90

The possibility that modest elevations in the level of blood homocysteine (hyperhomocysteinaemia) could contribute to cardiovascular disease arose from investigation of patients with rare, severe homocysteine elevations caused by cystathionine beta-synthase deficiency. Such patients often had thromboembolic events before the age of 30 years. Since the established cardiovascular risk factors could only partly account for the occurrence and severity of vascular disease in the general population, other risk factors had to exist, and homocysteine elevation seemed to be a possible candidate. Australian case-control studies identified an association between mild homocysteine elevation and early-onset coronary disease, and also with chronic renal failure. Patients in the latter group have a high prevalence of unexplained vascular disease and particularly high homocysteine levels. Such elevations in levels of homocysteine in vascular patients could usually be normalised by daily supplementation with folic acid (1-5 mg) while in patients with chronic renal failure 5 mg of folic acid daily markedly reduced the increased concentrations of homocysteine. These initial observations have been confirmed by many investigators and biologically plausible mechanisms for homocysteine-induced vascular dysfunction, and particularly endothelial dysfunction, have been identified. However, associations between hyperhomocysteinaemia and other risk factors, such as smoking and hypertension, have also been documented and need to be controlled for when assessing any increase in risk that homocysteine may independently confer. Although it has been established that lowering the greatly elevated blood homocysteine levels in homocystinuria, due to cystathione beta-synthase deficiency, unquestionably reduces cardiovascular risk, it remains to be determined whether normalising mild homocysteine elevation could reduce cardiovascular risk. Trials to test this possibility have been initiated and others are planned.
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PMID:Novel risk factors for vascular disease: the homocysteine hypothesis of cardiovascular disease. 991 68

Cystathionine beta-synthase (CBS), a pyridoxal 5'-phosphate (PLP) dependent enzyme, catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS was recently shown to be a heme protein. While the role of heme in CBS is unknown, catalysis by CBS can be explained solely by participation of PLP in the reaction mechanism. In this study, treatment of CBS with sodium borohydride selectively reduced the Schiff base but did not affect the heme. Purification and sequencing of the PLP-cross-linked peptide from a trypsin digest of the reduced enzyme revealed the evolutionarily conserved Lys119 to be the residue forming the Schiff base. Serine and hydroxylamine form an alpha-aminoacrylate and an oxime with PLP in CBS, respectively. The sulfhydryl-containing substrate, homocysteine, disturbs the heme environment but does not interact with PLP. In contrast to other PLP-dependent enzymes, CBS emits no PLP-related fluorescence when excited at 296 or 330 nm. PLP but not heme dissociates from the enzyme in the presence of hydroxylamine. The dissociation of PLP is a multistage process involving a short approximately 500 s lag phase, followed by a rapid inactivation and a slower PLP-oxime formation. PLP-free CBS exhibits a decrease of secondary structure as well as loss of CBS activity that can be only partially restored by PLP. This study constitutes the first comprehensive investigation of PLP interaction with a heme protein.
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PMID:Binding of pyridoxal 5'-phosphate to the heme protein human cystathionine beta-synthase. 1005 42

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


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