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Results for “Multifunctional Enzymes”

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PubMed1996

Expression of catalytically active human multifunctional glycogen-debranching enzyme and lysosomal acid alpha-glucosidase in insect cells.

Glycogen debranching enzyme and acid alpha-glucosdase are responsible for glycogen degradation in human. The formal enzyme is a multifunctional enzyme with two independent catalytic activities occurring on a single polypeptide, while the latter is a lysosomal enzyme which matures through extensive glycosylation and phosphorylation and proteolytic processing. Deficiency of glycogen debranching enzyme and acid alpha-glucosidase cause glycogen storage disease type III and II, respectively. Baculovirus/insect expression system was used to produce both GDE and GAA. Both enzymes were found to be catalytically and antigenically active. The majority of recombinant GDE is present in the medium (70%). Uptake experiment indicated that GAA produced in the insect cells could not be absorbed into the GSD type II patient fibroblasts through mannose-6-phosphate receptor mediated endocytosis. Uptake experiment combined with immunoblot analysis indicated there are differences in the posttranslational modification and processing between insect cells and mammalian cells.

Animals↗

PubMed2000

Unusual arrangement of catalytic domains in head-to-tail associated homodimer of 6-hydroxymellein synthase, a multifunctional polyketide biosynthetic enzyme.

6-Hydroxymellein synthase, a multifunctional polyketide synthetic enzyme in carrot, is organized as a homodimer, and the activity of the synthase was appreciably inhibited upon the specific alkylation of cysteine- and cysteamine-SHs at the reaction center with iodoacetoamide and chloroacetyl-CoA, respectively. Dissociation and stoichiometric recombination of the unmodified and the SH-modified enzyme subunits yielded a combination of unmodified-unmodified, unmodified-modified and modified-modified hybrid dimers that together exhibit 50% activity. In contrast, hybrid dimers obtained by reconstruction of the two modified enzymes showed essentially no catalytic activity. These results suggest that the two subunits of 6-hydroxymellein synthase are aligned in head-to-tail orientation to organize two reaction centers which are comprised of a cysteine and a complementary cysteamine SH group, belonging to and contributed from the same subunit in the homodimer structure.

Journal Article↗

PubMed1990

Isolation and characterization of the gene encoding the muscle-specific isozyme of human phosphoglycerate mutase.

The human muscle-specific phosphoglycerate mutase encoding gene (PGAM-M) has been cloned from a genomic cosmid library and sequenced. The sequence corresponding to the coding region was evaluated and revised by sequencing of the protein itself, fully confirming our results. The amino acid sequence of the M isozyme presented a 80.6% homology with the B isozyme (non-muscle-specific isozyme), a value higher than previously reported. The PGAM-M gene is composed of three exons, which consist of 454, 180 and 202 bp, respectively, and are separated by two introns of 103 bp and approx. 5.6 kb, respectively. Comparison of the structure of the human PGAM-M gene with that coding for human bisphosphoglycerate mutase, an erythroid-specific enzyme belonging to the same multifunctional enzyme family, revealed that the location of the second intron is similar in each gene and corresponds to a tertiary subdomain in the spatial structure of the protein. The transcription start point (tsp) in the PGAM-M gene was identified by both primer extension and S1 nuclease-protection experiments. A TATA-box-like element was observed 29 bp upstream from the tsp; the sequence ATTGG, inverse/complementary to CCAAT-box, was found 40 bp upstream from the supposed TATA box. No muscle-specific consensus sequences could be detected in the 5'-untranslated region. Only one polyadenylation AATAAA signal was observed in the short 3'-untranslated region (43 bp long). Finally, only one copy of this gene is present in the human genome instead of the several copies found for the PGAM-B gene, suggesting the possible evolutionary origin of the muscle subunit in a modified copy of the PGAM-B gene.

Amino Acid Sequence↗

PubMed1997

Copurification of dihydroxyacetone-phosphate acyl-transferase and other peroxisomal proteins from liver of fenofibrate-treated rats.

Dihydroxyacetone-phosphate acyl-transferase (DHAP-AT), a peroxisomal membrane-bound enzyme that catalyzes the first step of ether-glycerolipid synthesis, was purified from liver of rats treated with fenofibrate, a peroxisome proliferator. The protocol first included isolation of peroxisomes, their purification through a discontinuous gradient and solubilization of membranes in CHAPS. DHAP-AT was further purified by four chromatographic steps, namely low-pressure size-exclusion, cation-exchange, hydroxylapatite and chromatofocusing. The chromatofocusing step led to a 4000-fold increase in the specific activity of DHAP-AT with respect to the liver homogenate with a yield of about 0.2%. Trypsin digestion of a 64-kDa protein band upon SDS-PAGE resulted in a peptide sequence unknown in databases. A corresponding degenerated oligonucleotide was used as a probe in Northern blotting, and a transcript of 3.3 kb was detected in some rat tissues. Moreover, the overall procedure allowed co-purification of four major peroxisomal enzymes: urate-oxidase, catalase, multifunctional enzyme and palmitoyl-CoA oxidase, respectively.

Acyltransferases↗

PubMedDate not supplied

Genetic-dependency of peroxisomal cell functions - emerging aspects.

This paper reviews aspects concerning the genetic regulation of the expression of the well studied peroxisomal genes including those of fatty acid beta-oxidation enzymes; acyl-CoA oxidase, multifunctional enzyme and thiolase from different tissues and species. An important statement is PPARalpha, which is now long known to be in rodents the key nuclear receptor orchestrating liver peroxisome proliferation and enhanced peroxisomal beta-oxidation, does not appear to control so strongly in man the expression of genes involved in peroxisomal fatty acid beta-oxidation related enzymes. In this respect, the present review strengthens among others the emerging concept that, in the humans, the main genes whose expression is up-regulated by PPARalpha are mitochondrial and less peroxisomal genes. A special emphasis is also made on the animal cold adaptation and on need for sustained study of peroxisomal enzymes and genes; challenging that some essential roles of peroxisomes in cell function and regulation still remain to be discovered.

Adaptation, Biological↗

PubMed2000

The endonuclease activity of the yeast Dna2 enzyme is essential in vivo.

Dna2 is a multifunctional enzyme in yeast that possesses endonuclease activity well suited to remove RNA-DNA primers of Okazaki fragments, raising the question of whether endonuclease activity is essential for in vivo Dna2 function. Systematic site-directed mutations of amino acid residues in Saccharomyces cerevisiae DNA2 conserved in the central region of many eukaryotic DNA2 homologs allowed us to identify mutant dna2 alleles that were divided into three groups based on the viability of the mutant cells: (i) viable; (ii) inviable only when expression was repressed; (iii) inviable. Biochemical analyses of recombinant mutant Dna2 proteins isolated from the latter two groups revealed that they possessed normal ATPase/helicase activity, but were impaired in their endonuclease activity. Cells expressing mutant Dna2 enzymes partially impaired in endonuclease activity were viable, but were unable to grow when expression of their mutant Dna2 enzymes was further reduced. Their growth was restored when the mutant Dna2 proteins decreased in nuclease activity were induced to overexpress. In contrast, mutant Dna2 proteins lacking endonuclease activity did not allow cells to grow under any conditions tested. These in vivo and in vitro results demonstrate that the endonuclease activity of Dna2 is essential for Okazaki fragment processing.

Adenosine Triphosphatases↗

PubMed1996

Human glycogen debranching enzyme gene (AGL): complete structural organization and characterization of the 5' flanking region.

Glycogen debranching enzyme (gene symbol, AGL) is a multifunctional enzyme acting as 1,4-alpha-D-glucan:1,4-alpha-D-glucan 4-alpha-D-glycosyltransferase and amylo-1,6-glucosidase in glycogen degradation. Genetic deficiency of AGL activity causes glycogen storage disease type III (GSD-III). To determine the molecular basis of GSD-III and elucidate the mechanisms for controlling tissue-specific gene expression, we report the isolation and structural organization of the human chromosomal AGL gene. The gene is 85 kb in length and is composed of 35 exons, encoding a 7.0-kb mRNA. The first 2 exons and 68 bp of exon 3 contain 5' untranslated region. Translation begins in exon 3, which encodes the first 27 amino acids of the AGL. Exons 4 to 35 encode the remaining 1505 amino acids. Among the 6 isoforms identified, the major isoform (isoform 1) starts with exon 1 and is widely expressed, including expression in both liver and muscle. Muscle-specific isoforms (2, 3, and 4) begin with exon 2. Isoforms 5 and 6 are minor isoforms that begin further within the gene. Reporter assays revealed that promoter region 1 (for isoform 1) was functional in liver (HepG2 cells), muscle (C2C12 cells), and ovary (Chinese hamster ovary cells), and promoter region 2 (for muscle-specific isoforms) was active only in muscle. These results suggest that the human AGL gene contains at least 2 promoter regions that confer differential expression of isoform mRNAs in a tissue-specific manner.

Animals↗

PubMed2005

The evolution of connectivity in metabolic networks.

Processes in living cells are the result of interactions between biochemical compounds in highly complex biochemical networks. It is a major challenge in biology to understand causes and consequences of the specific design of these networks. A characteristic design feature of metabolic networks is the presence of hub metabolites such as ATP or NADH that are involved in a high number of reactions. To study the emergence of hub metabolites, we implemented computer simulations of a widely accepted scenario for the evolution of metabolic networks. Our simulations indicate that metabolic networks with a large number of highly specialized enzymes may evolve from a few multifunctional enzymes. During this process, enzymes duplicate and specialize, leading to a loss of biochemical reactions and intermediary metabolites. Complex features of metabolic networks such as the presence of hubs may result from selection of growth rate if essential biochemical mechanisms are considered. Specifically, our simulations indicate that group transfer reactions are essential for the emergence of hubs.

Biomass↗

PubMed2000

Protein crosslinking in assembly and remodelling of extracellular matrices: the role of transglutaminases.

Transglutaminases form a family of proteins that have evolved for specialized functions such as protein crosslinking in haemostasis, semen coagulation, or keratinocyte cornified envelope formation. In contrast to the other members of this protein family, tissue transglutaminase is a multifunctional enzyme apparently involved in very disparate biological processes. By virtue of its reciprocal Ca2+-dependent crosslinking activity or GTP-dependent signal transducing activity, tissue transglutaminase exhibits true multifunctionality at the molecular level. The crosslinking activity can subserve disparate biological phenomena depending on the location of the target proteins. Intracellular activation of tissue transglutaminase can give rise to crosslinked protein envelopes in apoptotic cells, whereas extracellular activation contributes to stabilization of the extracellular matrix and promotes cell-substrate interaction. While tissue transglutaminase synthesis and activation is normally part of a protective cellular response contributing to tissue homeostasis, the enzyme has also been implicated in a number of pathological conditions including fibrosis, atherosclerosis, neurodegenerative diseases, celiac disease, and cancer metastasis. This review discusses the role of transglutaminases in extracellular matrix crosslinking with a focus on the multifunctional enzyme tissue transglutaminase.

Animals↗

PubMed1995

Differential cellular and subcellular expression of the human multifunctional apurinic/apyrimidinic endonuclease (APE/ref-1) DNA repair enzyme.

The multifunctional mammalian apurinic/apyrimidinic endonuclease (APE) is responsible for the repair of apurinic/apyrimidinic sites in DNA. In addition, this enzyme has been shown to function as a redox factor facilitating the DNA-binding capability of JUN and FOS, as well as numerous other transcription factors through the alteration of the transcription factor redox state. Biochemical studies of organ homogenates have shown that APE is present in the different tissues studied at similar concentrations. The present study examines the immunohistochemical distribution of APE in several organs and demonstrates new and unexpected patterns of cellular and subcellular localization of this enzyme. In the hippocampus, the APE protein was highly expressed in neurons of the dentate gyrus and regions CA3 and CA4, and unexpectedly, the staining was primarily cytoplasmic. AP endonuclease immunoreactivity in the cerebellum was found in the granule and Purkinje cells, both cytoplasmic and nuclear. APE staining of the hypoglossal nucleus of the brainstem, where motor neurons that control tongue movement reside, showed reactivity in the cytoplasmic Nissl substance. Skin, liver, and duodenum demonstrated nuclear staining; however, in the duodenum, only the enterocyte nuclei of the proximal villus and the crypts of Lieberkuhn were stained, with no staining of the distal villus. These results suggest that APE has different regulatory and functional roles in different cells and organs of the body. This study shows the importance of correlating in vitro findings in tissue culture cells with the organism as a whole. The cytoplasmic staining seen in parts of the brain and in liver suggests that there may be additional functions for the APE yet to be described.

Autopsy↗

PubMed1987

Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis.

An approximately 16-kilobase pair region of the Bacillus subtilis chromosome at 55 degrees containing genes for de novo purine nucleotide synthesis (Piggot, P. J., and Hoch, J. A. (1985) Microbiol. Rev. 49, 158-179) was cloned. The nucleotide sequence of over 13 kilobase pairs indicates that this region contains a cluster of 12 genes, 11 of which encode enzymes that catalyze the 10 reactions for de novo purine nucleotide synthesis from 5-phosphoribosyl 1-pyrophosphate to IMP. The genes were identified by complementation of Escherichia coli pur mutants and by sequence comparisons with homologous enzymes. The cluster is likely an operon and is organized into three groups of overlapping genes followed by the last gene: purEKB-purC(orf)QLF-purMNH(J)-purD. Sequence comparisons provide evidence for homology of monofunctional purine nucleotide biosynthetic enzymes from B. subtilis with the corresponding multifunctional enzymes from yeast and Drosophila. Sequence alignment of the phosphoribosylaminoimidazole carboxylase heterodimer from B. subtilis with the monomeric enzyme from Methanobrevibacter smithii indicates an evolutionary relationship between these two enzymes. S1 nuclease analysis was used to map the mRNA 5' and 3' ends and to estimate levels of mRNA. These experiments indicate that synthesis of purine nucleotides is regulated independently by adenine and guanine nucleotides. Adenine nucleotides regulate transcription initiation. Guanine nucleotides regulate transcription by a termination-antitermination mechanism in a 242-nucleotide 5' untranslated mRNA leader region. Groups of overlapping genes, regulated at least in part by transcription termination-antitermination is likely to be a common theme for genetic organization and regulation of biosynthetic genes in this Gram-positive organism.

Amino Acid Sequence↗

PubMed1985

C1-Tetrahydrofolate synthase from rabbit liver. Structural and kinetic properties of the enzyme and its two domains.

C1-Tetrahydrofolate synthase is a multifunctional enzyme which catalyzes three reactions in 1-carbon metabolism: 10-formyltetrahydrofolate synthetase; 5,10-methenyltetrahydrofolate cyclohydrolase; 5,10-methylenetetrahydrofolate dehydrogenase. A rapid 1-day purification procedure has been developed which gives 40 mg of pure enzyme from 10 rabbit livers. The 10-formyltetrahydrofolate synthetase activity of this trifunctional enzyme has a specific activity that is 4-fold higher than the enzyme previously purified from rabbit liver. Conditions have been developed for the rapid isolation of a tryptic fragment of the enzyme which contains the methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities. This fragment is a monomer exhibiting a subunit and native molecular weight of 36,000 in most buffers. However, in phosphate buffers the native molecular weight suggests that the fragment is a dimer. Conditions are also given whereby chymotryptic digestion allows the simultaneous isolation from the native enzyme of a large fragment containing the 10-formyltetrahydrofolate synthetase activity and a smaller fragment containing the dehydrogenase and cyclohydrolase activities. The large fragment is a dimer with a subunit molecular weight of 66,000. The small fragment retains all of the dehydrogenase and cyclohydrolase activities of the native enzyme. The large fragment is unstable but retains most of the 10-formyltetrahydrofolate synthetase activity. Km values of substrates for the two fragments are the same as the values for the native enzyme. The 10-formyltetrahydrofolate synthetase activity of the native enzyme requires ammonium or potassium ions for expression of full catalytic activity. The effect of these two ions on the catalytic activity of the large chymotryptic fragment is the same as with the native enzyme. We have shown by differential scanning calorimetry that the native enzyme contains two protein domains which show thermal transitions at 47 and 60 degrees C. Evidence is presented that the two domains are related to the two protein fragments generated by proteolysis of the native enzyme. The larger of the two domains contains the active site for the 10-formyltetrahydrofolate synthetase activity while the smaller domain contains the active site which catalyzes the dehydrogenase and cyclohydrolase reactions. Replacement of sodium ion buffers with either ammonium or potassium ions results in an increase in stability of the large domain of the native enzyme. This change in stability is not accompanied by a change in the quaternary structure of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminohydrolases↗

PubMed1985

Fatty acid biosynthesis in Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin. Purification and characterization of a novel fatty acid synthase, mycocerosic acid synthase, which elongates n-fatty acyl-CoA with methylmalonyl-CoA.

A crude extract from Mycobacterium tuberculosis var. bovis Bacillus Calmette-Guérin was previously shown to incorporate methylmalonyl-CoA into mycocerosic acids, exemplified by 2,4,6,8-tetramethyloctacosanoic acid, and malonyl-CoA into n-fatty acids (Rainwater D. L., and Kolattukudy, P. E. (1983) J. Biol. Chem. 258, 2979-2985). The presence of several fatty acid synthases with differences in substrate preference and product chain length was detected in the crude extract of M. tuberculosis var. bovis. Among them was a mycocerosic acid synthase which was purified to homogeneity using anion-exchange chromatography, gel filtration, affinity chromatography, and hydroxylapatite chromatography. This fatty acid synthase elongated long-chain fatty acyl-CoA primers using methylmalonyl-CoA and NADPH to produce multimethyl-branched mycocerosic acids. The enzyme was specific for methylmalonyl-CoA and would not incorporate malonyl-CoA into fatty acids. It elongated n-C6 to n-C20 CoA esters to generate primarily the corresponding tetramethyl-branched mycocerosic acids. Exogenous [1-14C]acyl-CoA and trideuteromethylmalonyl-CoA were incorporated into the multimethyl-branched fatty acids. Dodecyl sulfate electrophoresis showed that the enzyme had a molecular weight of 238,000, whereas gel filtration showed a native molecular weight of 490,000, indicating that the enzyme is composed of two monomers of identical molecular weight. The enzyme contained an acyl carrier protein-like segment as indicated by incorporation of [1-14C] pantothenate into the 238-kDa protein and production of 1 mol of taurine/mol of the monomer upon hydrolysis of performic acid-oxidized enzyme. It is concluded that the mycocerosic acid synthase is a multifunctional enzyme similar to the well-characterized multifunctional fatty acid synthases except for the substrate specificity.

Acyl Coenzyme A↗

PubMed1984

Evolution of catalytic proteins or on the origin of enzyme species by means of natural selection.

It is believed that all present-day organisms descended from a common cellular ancestor. Such a cell must have evolved from more primitive and simpler precursors, but neither their organization nor the route such evolution took are accessible to the molecular techniques available today. We propose a mechanism, based on functional properties of enzymes and the kinetics of growth, which allows us to reconstruct the general course of early enzyme evolution. A precursor cell containing very few multifunctional enzymes with low catalytic activities is shown to lead inevitably to descendants with a large number of differentiated monofunctional enzymes with high turnover numbers. Mutation and natural selection for faster growth are shown to be the only conditions necessary for such a change to have occurred.

Biological Evolution↗

PubMed1989

Interaction of folylpolyglutamates with enzymes in one-carbon metabolism.

Of all the coenzymes, tetrahydrofolate exhibits the most structural diversity. The relationship of these structural forms to physiological function is under intense study by numerous research groups. In textbooks, tetrahydrofolate (tetrahydropteroylmonoglutamate) is shown as the coenzyme of one-carbon metabolism, but it has been known for several decades that the physiologically active forms of the coenzyme contain from 4 to 7 glutamyl residues linked by amide bonds through the gamma-carboxyl group. These glutamyl residues do not serve a direct function in transferring the one-carbon group. The tetrahydrofolylpolyglutamates were originally thought to be simply storage forms of the coenzyme, but studies now show that the polyglutamate chain of the coenzyme affects the transport properties of the coenzyme, alters the kinetic properties of many enzymes in one-carbon metabolism, and results in channeling of the coenzyme between several enzymes. In general, the dissociation constants of this group of enzymes for the tetrahydrofolylpolyglutamates are very low, in the 0.1 to 1 microM range. The concentration of the coenzyme in the cell appears to be similar to the concentration of folate-utilizing enzymes, suggesting that the concentration of unbound coenzyme in the cell may be very low. Several of the enzymes in one-carbon metabolism are either multifunctional proteins or multienzyme complexes. An active area of research is to determine if there is a functional relationship between these multifunctional enzymes and the polyglutamate portion of the coenzyme.

Carbon↗

PubMed2005

[Lysosomal carboxypeptidase A].

Lysosomal carboxypeptidase A (cathepsin A) is synthetized in the form of preproenzyme, which undergoes to active enzyme as a result of post-translational modification. It splits off C-terminal amino acid residues from peptides and proteins and synergizes with other proteases in degradation of cellular proteins in lysosomes. Lysosomal carboxypeptidase A has an effect on peptide hormones and peptides of biological activity of tissues and body fluids as well. It forms complexes with some glycosidases that protects them against proteolytic degradation. Deficiency of this enzyme induces storage diseases. Lysosomal carboxypeptidase A as multifunctional enzyme plays an important regulatory role in organismal metabolism.

Amino Acid Sequence↗

PubMed1993

Hematopoietic growth factor induction of gamma-glutamyl transferase in the KG-1 myeloid cell line.

The enzyme gamma-glutamyl transferase (GGT) is a multifunctional enzyme that participates in a number of metabolic processes, including the conversion of leukotriene C4(LTC4) to leukotriene D4(LTD4). LTD4 is necessary for normal myeloid proliferation and differentiation. We have examined the ability of hematopoietic growth factors (HGF) to induce GGT enzyme activity and mRNA content in a HGF-responsive cell line (KG-1). Incubation of KG-1 with recombinant human cytokines interleukin-1 beta (IL-1 beta), interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF), but not interleukin-6 (IL-6), granulocyte colony-stimulating factor (G-CSF) or monocyte colony-stimulating factor (M-CSF), results in significant increases in GGT enzyme activity. The increases in GGT activity are both dose- and time-dependent. In response to IL-1, increases in enzyme activity are seen by 6 hours and activity is maximal by 24 hours. GGT mRNA increases also occur and peak by 3 to 6 hours. These results indicate that induction of increases in GGT mRNA levels and enzyme activity occur in myeloid cells in response to HGFs. This induction, together with the requirement for LTD4 for normal granulopoiesis, supports a role for GGT in the cellular events occurring in myeloid cells in response to HGFs.

Antibodies↗

PubMed1998

Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate.

Fatty acid synthase is an important enzyme participating in energy metabolism in vivo. The inactivation and conformational changes of the multifunctional fatty acid synthase from chicken liver in SDS solutions have been studied. The results show that the denaturation of this multifunctional enzyme by SDS occurred in three stages. At low concentrations of SDS (less than 0.15 mM) the enzyme was completely inactivated with regard to the overall reaction. For each component of the enzyme, the loss of activity occurred at higher concentrations of SDS. Significant conformational changes (as indicated by the changes of the intrinsic fluorescence emission and the ultraviolet difference spectra) occurred at higher concentrations of SDS. Increasing the SDS concentration caused only slight changes of the CD spectra, indicating that SDS had no significant effect on the secondary structure of the enzyme. The results suggest that the active sites of the multifunctional fatty acid synthase display more conformational flexibility than the enzyme molecule as a whole.

Animals↗