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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)

We have developed conditions for efficient cDNA cloning of nanogram amounts of purified mRNAs coding for cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and for the cytosolic precursors of mitochondrial ornithine transcarbamylase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) and the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.3]. The three mRNAs, prepared by sequential immunoselection from the same batch of rat liver polysomes, were pooled (20 ng each), and cDNA was synthesized by using avian reverse transcriptase. The second DNA strand was prepared by "nick-translation repair" of the cDNA . mRNA hybrid with RNase H, polymerase I, and DNA ligase from Escherichia coli. The double-stranded (ds) DNA was tailed with deoxycytidine residues, annealed with Pst I-cut/dG-tailed pBR322, and used to transform E. coli. The library generated by this three-step procedure contained 5000 independent colonies. A 550-base-pair (bp) cDNA clone of the beta subunit of propionyl-CoA carboxylase was detected by hybrid-selected translation; it was then used to screen the library for longer cDNAs. Two hybridizing cDNAs, 1200 and 1000 bp long with a 200-bp overlap, representing together a full-length copy of the coding region and 446 bp of 3' untranslated sequence, were recovered. Each plasmid mapped to the region q13.3----q22 of human chromosome 3. Cystathionine beta-synthase clones were obtained by screening the library with a single-stranded [32P]cDNA prepared directly from the highly purified synthase mRNA by reverse transcriptase. The longest hybridizing cDNA of 1700 bp was used in hybrid-selected translation and detected a polypeptide of 63 kDa, identical in size to rat liver synthase. In situ hybridization of this cDNA to q22 of human chromosome 21 confirmed two previous tentative assignments of the synthase locus to this chromosome.
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PMID:Cloning and screening with nanogram amounts of immunopurified mRNAs: cDNA cloning and chromosomal mapping of cystathionine beta-synthase and the beta subunit of propionyl-CoA carboxylase. 345 73

It has been shown that yeast tryptophan synthase (L-serine hydro-lyase (adding indoleglycerol-phosphate) EC 4.2.1.20) catalyses tritium exchange reactions between protons on the alpha-carbon of L-serine of L-tryptophan, and water. The absolute rates of these reactions and indole-serine condensation (reaction B), all of which are pyridoxal phosphate-dependent, were measured. L-Serine exchange was resolved into two components, a high-affinity, slow, Michaelian reaction (KmS,H = 0.06 mM, kcats,H 3 X 10(-3) s-1) and a faster reaction (kcat greater than 2.5 S-1) which was not saturated even at 100 mM L-serine. Hydrogen exchange by tryptophan was a Michaelian process (KmT,H = 2.9 mM; kcatT,H = 0.6 s-1). Indole did not inhibit either exchange reaction. A plausible explanation of the results, that reaction B has a ping-pong mechanism with serine as first substrate and water and L-tryptophan as first and second products, respectively, was inadequate because of the observations that L-tryptophan is as first and second products, respectively, was inadequate because of the observations that L-tryptophan is synthesised with less than 1 mol of exchanged proton per mol amino acid, and that the ratio kcat/Km for serine changes between enzyme reactions. A branched modification with two enzyme-serine complexes, only one of which will exchange protons with water, will fit all the results.
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PMID:Hydrogen exchange kinetics and the mechanism of reaction B of yeast tryptophan synthase. 393 73

We have purified three low-abundance hepatic mRNAs to near homogeneity by polysome immunoadsorption. The mRNAs coding for the precursor of ornithine transcarbamoylase [carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3], the precursor of the beta-subunit of propionyl-CoA carboxylase [propionyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3], and cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], representing approximately 0.20, 0.02, and 0.015% of total hepatic mRNA, respectively, were purified 450- to 6,300-fold. We used the following steps: interaction of rat liver polysomes with an IgG fraction of monospecific antisera raised against each polypeptide; immobilization of polysome-antibody complexes on a protein A-Sepharose column; removal of the bulk of polysomes by extensive washing; dissociation of ribosomal subunits and elution of specific mRNA with EDTA; and isolation of the eluted mRNA by chromatography on an oligo(dT)-cellulose column. It seems likely that this procedure will permit isolation of other low-abundance mRNAs and subsequent cloning of their respective cDNAs.
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PMID:Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption. 618 Apr 33

The immunochemical reactivity of unfractionated antibodies elicited by denatured beta 2 subunits of Escherichia coli tryptophan synthase [L-serine hydro-lyase (adding indole) EC 4.2.1.20] with the homologous antigen and with the native enzyme is examined. These antibodies recognize the native apoenzyme nearly as well as the denatured protein. On the contrary, after binding of its cofactor, pyridoxal 5'-phosphate, the protein exhibits a much lower immunoreactivity toward these antibodies. This decrease of affinity becomes even more pronounced when the beta 2 protein interacts with the alpha subunit. Similarly, reduction of the Schiff base formed between the cofactor and the protein leads to a strong decrease of immunoreactivity. To account for these results, it is proposed that apo-beta 2 must be a dynamic flexible structure that easily exposes to the solvent regions of its polypeptide chain that normally are buried in its interior. The increase in rigidity of this structure upon binding of the cofactor, reduction of Schiff base, and formation of the alpha 2 beta 2 complex would then account for the decreased immunoreactivity of these various states of the native beta 2 protein.
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PMID:Immunochemical evidence for conformational flexibility and its modulation by specific ligands in the beta 2 subunit of Escherichia coli tryptophan synthase. 619 71

The previously published procedure (Kraus et al. (1978) J. Biol. Chem. 253, 6523-6528) for the purification of cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine) EC 4.2.1.22], a pyridoxal 5'-phosphate-dependent enzyme from human liver has been modified. The new procedure, starting with a liver homogenate "aged" for 7 days at 4 degrees C, yielded homogeneous enzyme purified over 3000-fold with a much improved yield. "Aging" of the enzyme in crude homogenates yields a form apparently smaller by gel electrophoresis and with significantly increased activity and antigenicity. This species of cystathionine beta-synthase does not form stable complexes with other proteins during purification as does the previously employed, freshly used species. An absorption spectrum and an amino acid composition of the pure enzyme were determined; the amino-terminal residue was shown to be methionine. The isoelectric points of holosynthase and aposynthase were estimated to be 5.2 and 5.6, respectively. Rabbit antiserum raised against the pure cystathionine beta-synthase was characterized using as antigen crude synthase from five different mammalian species as well as the pure human enzyme.
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PMID:Cystathionine beta-synthase from human liver: improved purification scheme and additional characterization of the enzyme in crude and pure form. 683 28

The activities of cystathionine synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], uroporphyrinogen I synthase [porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8], and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) have been measured in phytohemagglutinin-stimulated lymphocytes of young and old human subjects. A significant decrease in activity with age was observed for cystathionine synthase and uroporphyrinogen I synthase but not for glucose-6-phosphate dehydrogenase. These changes could not be related to declining phytohemagglutinin response with aging. Age-related decreases in activity of some enzymes may be relevant for an understanding of the biology of aging. False assignment of heterozygosity, and even homozygosity, for certain genetic disorders, such as homocystinuria, may result when low enzyme levels are detected in the lymphocytes of older people.
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PMID:Effect of chronologic age on induction of cystathionine synthase, uroporphyrinogen I synthase, and glucose-6-phosphate dehydrogenase activities in lymphocytes. 694 Jan 98

In order to ascertain the role of L-serine sulfhydro-lyase (L-serine hydro-lyase (adding homocysteine) EC 4.2.1.22) which also catalyzes sulfhydrylation of O-acetyl-L-serine (Yamagata, S. (1981) J. Bacteriol. 147, 688-690), the enzyme was partially purified from a wild-type strain and three cysteine auxotrophs of Saccharomyces cerevisiae, and the molecular and enzymatic properties of these preparations were compared. The results showed no significant difference in properties investigated, indicating that cysteine synthesis is exclusively performed in this organism through sulfhydrylation of O-acetyl-L-serine, catalyzed not by serine sulfhydro-lyase but by O-acetylserine . O-acetylhomoserine sulfhydro-lyase (Yamagata, S., Takeshima, K. and Naiki, N. (1974) J. Biochem. 75, 1221-1229). Insensitivity of the former enzyme to L-methionine also supported this conclusion.
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PMID:Partial purification and comparison of some properties of L-serine sulfhydro-lyase of Saccharomyces cerevisiae. 703 82

Pyridoxal-5'-phosphate-dependent enzymes catalyze manifold reactions in the metabolism of amino acids. A comprehensive comparison of amino acid sequences has shown that most of these enzymes can be assigned to one of three different families of homologous proteins. The sequences of the enzymes of each family were aligned and their homology confirmed by profile analysis. Scrutiny of the reactions catalyzed by the enzymes showed that their affiliation with one of the three structurally defined families correlates in most cases with their regio-specificity. In the largest family, the covalency changes of the substrate occur at the same carbon atom that carries the amino group forming the imine linkage with the coenzyme. This family was thus named alpha family. It comprises glycine hydroxymethyltransferase, glycine C-acetyltransferase, 5-aminolevulinate synthase, 8-amino-7-oxononanoate synthase, all aminotransferases (with the possible exception of subgroup III), a number of other enzymes relatively closely related with the aminotransferases and very likely a certain group of amino acid decarboxylases as well as tryptophanase and tyrosine phenol-lyase which, however, catalyze beta-elimination reactions. The beta family includes L- and D-serine dehydratase, threonine dehydratase, the beta subunit of tryptophan synthase, threonine synthase and cysteine synthase. These enzymes catalyze beta-replacement or beta-elimination reactions. The gamma family incorporates O-succinylhomoserine (thiol-lyase, O-acetylhomoserine (thiol)-lyase, and cystathionine gamma-lyase, which catalyze gamma-replacement or gamma-elimination reactions, as well as cystathionine beta-lyase. The alpha and gamma family might be distantly related with one another, but are clearly not homologous with the beta family. Apparently, the primordial pyridoxal-5'-phosphate-dependent enzymes were regio-specific catalysts, which first specialized for reaction specificity and then for substrate specificity. The following pyridoxal-5'-phosphate-dependent enzymes seem to be unrelated with the alpha, beta or gamma family by the criterion of profile analysis:alanine racemase, selenocysteine synthase, and many amino acid decarboxylases. These enzymes may represent yet other families of B6 enzymes.
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PMID:Evolutionary relationships among pyridoxal-5'-phosphate-dependent enzymes. Regio-specific alpha, beta and gamma families. 811 47

Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.
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PMID:Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein. 1076 67

Cystathionine beta-synthase [CBS; L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] catalyzes the first committed step of transsulfuration in both yeast and humans. It has been established previously that human CBS is a hemeprotein but although the heme group appears to be essential for CBS activity, the exact function of the heme group is unknown. CBS activity is absent in heme deficient strains of Saccharomyces cerevisiae grown without heme supplementation. CBS activity can be restored by supplementing these strains with heme, implying that there is a heme requirement for yeast CBS. We subcloned, overexpressed and purified yeast CBS. The yeast enzyme shows absolute pyridoxal 5'-phosphate (PLP) dependence for activity but we could find no evidence for the presence of a heme group. Given the degree of sequence and mechanistic similarity between yeast and human CBS, this result indicates that heme is unlikely to play a direct catalytic role in the human CBS reaction mechanism. Further characterization revealed that, in contrast to human CBS, S-adenosylmethionine (AdoMet) does not activate yeast CBS. Yeast CBS was found to be coordinately regulated with proliferation in S. cerevisiae. This finding is the most likely explanation of the observed apparent heme dependence of transsulfuration in vivo.
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PMID:Transsulfuration in Saccharomyces cerevisiae is not dependent on heme: purification and characterization of recombinant yeast cystathionine beta-synthase. 1105 61


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