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PubMed1985

Metabolic pathways as enzyme complexes: evidence for the synthesis of phenylpropanoids and flavonoids on membrane associated enzyme complexes.

In earlier studies [G. Hrazdina, G. J. Wagner, and H. W. Siegelman (1978) Phytochemistry 17, 53-56; G. J. Wagner and G. Hrazdina (1984) Plant Physiol. 74, 901-906], evidence was obtained suggesting that the endoplasmic reticulum was a site for phenylpropanoid and flavonoid metabolism in petal tissue, and that (a) multienzyme complex(es) might be involved in this metabolism. Now, the possible role of membrane-bound multienzyme complexes in phenylpropanoid and flavonoid metabolism in three tissues has been investigated by (1) correlating enzyme induction kinetics and rates, (2) examining the molecular weight of putative complexes, (3) channeling of substrates, (4) determining the susceptibility of bound activities to trypsin digestion, and (5) investigating the structurally linked latency of bound activities. Results suggest that at least a part--and possibly the entire pathway--from phenylalanine to flavonoids is membrane (endoplasmic reticulum) associated, and that this metabolism is facilitated by a multienzyme complex. Phenylalanine ammonia lyase, the first enzyme of the biosynthetic sequence, and a flavonoid glucosyltransferase, the last, appear to be located in the lumen of the membranes. Cinnamate 4-hydroxylase is membrane embedded, while other enzyme activities appear to be weakly associated with the cytoplasmic face of endoplasmic reticulum membranes.

Anthocyanins↗

PubMedDate not supplied

[Principles of integration of cell metabolism].

The notion of the "primary blocks" of cellular metabolism (designated as "metabolic system") has been introduced. Metabolic system is defined as a metabolic pathway which corresponds to the really existing multienzyme complex. The complex of glycolytic enzymes which catalyzes the anaerobic reduction of glucose-6-phosphate with production of ATP may serve as an example of metabolic system (this complex does not contain hexokinase). The complex is formed on thin filaments of I-band of the muscle fibers or on dimers of band 3 protein embedded in the erythrocyte membranes. The fixation of the multienzyme complex to the support of biological nature provides the material basis for regulation of the metabolic system by chemical signals produced by the higher levels of metabolic control. Owing to interaction with anchor protein of the support the chemical signals exert the general control of functioning the multienzyme complex (switching on--switching-off of the metabolic system). It is assumed that the glycolytic system in skeletal muscles is stimulated by Ca2+ ions which interact with the anchor protein of the support (troponin C).

Allosteric Regulation↗

PubMed1990

Glutaminyl-tRNA synthetase as a component of the high-molecular weight complex of human aminoacyl-tRNA synthetases. An immunological study.

The human glutaminyl-tRNA synthetase is three times larger than the corresponding bacterial and twice as large as the yeast enzyme. It is possible that the additional sequences of the human glutaminyl-tRNA synthetase are required for the formation of the multienzyme complex which is known to include several of aminoacyl-tRNA synthetases in mammalian cells. To address this point we prepared antibodies against three regions of the human glutaminyl-tRNA synthetase, namely against its enzymatically important core region, and against two sections in its large C-terminal extension. In intact multienzyme complexes the core region was accessible to specific antibody binding. However, the C-terminal sections became available to specific antibody binding only when certain components of the multienzyme complex were either absent or degraded. These findings allow first conclusions as to the relative position of some components in the mammalian aminoacyl-tRNA synthetase complex.

Amino Acyl-tRNA Synthetases↗

PubMed2006

Paenibacillus curdlanolyticus strain B-6 xylanolytic-cellulolytic enzyme system that degrades insoluble polysaccharides.

A facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6, isolated from an anaerobic digester produces an extracellular xylanolytic-cellulolytic enzyme system containing xylanase, beta-xylosidase, arabinofuranosidase, acetyl esterase, mannanase, carboxymethyl cellulase (CMCase), avicelase, cellobiohydrolase, beta-glucosidase, amylase, and chitinase when grown on xylan under aerobic conditions. During growth on xylan, the bacterial cells were found to adhere to xylan from the early exponential growth phase to the late stationary growth phase. Scanning electron microscopic analysis revealed the adhesion of cells to xylan. The crude enzyme preparation was found to be capable of binding to insoluble xylan and Avicel. The xylanolytic-cellulolytic enzyme system efficiently hydrolyzed insoluble xylan, Avicel, and corn hulls to soluble sugars that were exclusively xylose and glucose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of a crude enzyme preparation exhibited at least 17 proteins, and zymograms revealed multiple xylanases and cellulases containing 12 xylanases and 9 CMCases. The cellulose-binding proteins, which are mainly in a multienzyme complex, were isolated from the crude enzyme preparation by affinity purification on cellulose. This showed nine proteins by SDS-PAGE and eight xylanases and six CMCases on zymograms. Sephacryl S-300 gel filtration showed that the cellulose-binding proteins consisted of two multienzyme complexes with molecular masses of 1,450 and 400 kDa. The results indicated that the xylanolytic-cellulolytic enzyme system of this bacterium exists as multienzyme complexes.

Bacteria, Anaerobic↗

PubMed1983

Gel filtration of a complex of DNA polymerase and DNA precursor-synthesizing enzymes from a human lymphoblastoid cell line.

A multienzyme complex containing at least DNA polymerase (EC 2.7.7.7), thymidine kinase (EC 2.7.1.21), dTMP kinase (EC 2.7.4.9) nucleoside diphosphokinase (EC 2.7.4.6) and thymidylate synthetase was separated from the corresponding free enzymes of DNA precursor synthesis by gel filtration of a gently lysed preparation of HPB-ALL cells (a human lymphoblastoid cell line). The isolated incorporated the distal DNA precursors [3H]thymidine or [3H]dTMP into an added DNA template at rates comparable to those observed using the immediate precursor [3H]dTTP. Measurement of the apparent overall concentrations of [3H]dTTP produced during incorporation of [3H]thymidine and of [3H]dTMP were so low as to suggest that these precursors were channelled into DNA by the operation of a kinetically linked complex of precursor-synthesizing enzymes and of DNA polymerase. The DNA polymerase inhibitor 1-beta-D-arabinofuranosylcytosine triphosphate reduced incorporation of distal precursors into DNA. However [3H]dTTP did not accumulate in the reaction mixture. This suggested that the DNA polymerase regulated the flow of substrates through the complex. The results in this paper constitute direct evidence for the existence of multienzyme complexes of DNA synthesis in mammalian cells.

Cell Line↗

PubMed1980

[Hydrolysis of microcrystalline cellulose by multienzyme cellulase complexes of various origins].

The kinetic regularities of glucose and cellobiose formation from microcrystalline cellulose (MCC) under the action of cellulase complexes from eight different sources were studied. By means of successive addition of selected components of the cellulase complexes (endoglucanase and cellobiase) to the reaction system the rate-limiting steps for multienzymatic hydrolysis of MCC were determined. It was shown that in most cases the rate-limiting step of glucose formation (via hydrolysis of the intermediate cellobiose) is the cellobiase action. In a single case only (with a cellulase complex from Aspergillus foetidus enriched with cellobiase) the rate of glucose formation from MCC was limited by the endoglucanase action. In accordance with the kinetic theory developed it was shown that the addition of cellobiase excess to the reaction system resulted in changes of the rate-limiting step over to endoglucanase attack on the non-soluble cellulose for all cellulase complexes under study. Under the given experimental conditions a linear correlation between the steady-state ready of glucose formation from MCC under the action of all cellulase complexes on the on hand, and the endoglucanase activity of these complexes, on the other, was established. It was shown that the action of all cellulase (arbitrarily selected ones) is described by principally the same kinetic regularities, which, in turn, is indicative of identical mechanisms for hydrolysis of the insoluble cellulose under effects of cellulase complexes of various origin.

Actinomycetales↗

PubMed1997

Improvement of diffraction quality upon rehydration of dehydrated icosahedral Enterococcus faecalis pyruvate dehydrogenase core crystals.

Members of the family of 2-oxoacid dehydrogenase multienzyme complexes catalyze the oxidative decarboxylation of alpha-keto acids and are among the most remarkable enzymatic machineries in the living cell. These multienzyme complexes combine a highly symmetric (cubic or icosahedral) core with a dynamic and flexible arrangement of numerous subunits and domains surrounding the core. The center of the complex is formed by either 24 or 60 copies of dihydrolipoamide acetyltransferase (E2)-a multidomain enzyme. The hollow icosahedral cores are composed of 60 identical subunits of the catalytic domain of E2 with a molecular weight of about 1.8 million Da. Bipyramidal crystals suitable for X-ray diffraction of the icosahedral core of the pyruvate dehydrogenase multienzyme complex from Enterococcus faecalis were grown up to 0.7 mm in each dimension. The crystals belong to space group R32 with a = b = 244.3 A (hexagonal setting), and have a solvent content of 73%. The asymmetric unit contains one-third of the molecule, i.e., 20 of the 60 subunits. Initial X-ray crystallographic data to 7 A resolution were collected at cryotemperatures at synchrotron facilities. Interestingly, the diffraction was improved significantly upon rehydrating dehydrated crystals and extended to 4.2 A.

Crystallography, X-Ray↗

PubMedDate not supplied

[Symmetry-regulated dynamics of multi-enzyme complexes. A model of a pyruvate dehydrogenase complex from Escherichia coli].

A dynamic model for quaternary structure of a multienzyme complex is considered. The model is based on the supposition of simultaneously existing similar subunits in a number of different conformational states in the "core" of the multienzyme complex. It is supposed that cyclic conformational transitions of the "core" subunits conserve the symmetry of the entire complex. Such transitions drive the core dynamics as well as the suprastructural multienzyme dynamics. The dynamic model is constructed for the pyruvate dehydrogenase complex from E. coli in a supposition of three different conformers existing in its "core" which correspond to the three steps of the cyclic catalytic process. The model is in accordance with the data from the literature.

Acetylation↗

PubMed1997

Purification and characterization of a smooth muscle myosin light chain kinase-phosphatase complex.

We show that a myofibrillar form of smooth muscle myosin light chain phosphatase (MLCPase) forms a multienzyme complex with myosin light chain kinase (MLCKase). The stability of the complex was indicated by the copurification of MLCKase and MLCPase activities through multiple steps that included myofibril preparation, gel filtration chromatography, cation (SP-Sepharose BB) and anion (Q-Sepharose FF) exchange chromatography, and affinity purification on calmodulin and on thiophosphorylated regulatory light chain columns. In addition, the purified complex eluted as a single peak from a final gel filtration column in the presence of calmodulin (CaM). Because a similar MLCPase is present in varying amounts in standard preparations of both MLCKase and myosin filaments, we have named it a kinase- and myosin-associated protein phosphatase (KAMPPase). The KAMPPase multienzyme complex was composed of a 37-kDa catalytic (PC) subunit, a 67-kDa targeting (PT) subunit, and MLCKase with or without CaM. The approximate molar ratio of the PC and PT subunits was 1:2 with a variable and usually higher molar content of MLCKase. The targeting role of the PT subunit was directly demonstrated in binding experiments in which the PT subunit bound to both the kinase and to CaM. Its binding to CaM was, however, Ca2+-independent. MLCKase and the PT subunit potentiated activity of the PC subunit when intact myosin was used as the substrate. These data indicated that there is a Ca2+-independent interaction among the MLCPase, MLCKase, and CaM that are involved in the regulation of phosphatase activity.

Animals↗

PubMed1988

T4 phage deoxyribonucleoside triphosphate synthetase: purification of an enzyme complex and identification of gene products required for integrity.

We have isolated a highly enriched preparation of the multienzyme complex which synthesizes deoxyribonucleoside triphosphates (dNTPs) from bacteriophage T4-infected bacteria. By a combination of SDS polyacrylamide gel electrophoresis and assays for specific enzyme activities, we have been able to identify in our final preparation ten different gene products which were previously identified as constituents of this complex, based upon studies with crude preparations. The complex dissociates at high concentrations of NaCl and MgCl2 but is stable under ionic conditions thought to exist in vivo. The purified complex catalyzes the efficient five-step conversion of dCTP to dTTP. Experiments with several T4 mutants have demonstrated that gene products encoded by cd, regA, nrdA, and nrdB are necessary to retain physical integrity of the complex throughout the preparative procedure, while gp44, gp55, and gppseT are not required. We conclude from this evidence that the T4 early gene products which function in dNTP biosynthesis are, in fact, physically linked as a multienzyme complex, and that regA contributes to the integrity of this complex. However, the dNTP-synthesizing complex as we isolate it contains no detectable DNA polymerase, nor have other known replication proteins been detected.

DCMP Deaminase↗

PubMed1996

Association of N-acetylgalactosamine-6-sulfate sulfatase with the multienzyme lysosomal complex of beta-galactosidase, cathepsin A, and neuraminidase. Possible implication for intralysosomal catabolism of keratan sulfate.

N-Acetylgalactosamine-6-sulfate sulfatase (GALNS) catalyzes the first step of intralysosomal keratan sulfate (KS) catabolism. In Morquio type A syndrome GALNS deficiency causes the accumulation of KS in tissues and results in generalized skeletal dysplasia in affected patients. We show that in normal cells GALNS is in a 1.27-MDa complex with three other lysosomal hydrolases: beta-galactosidase, alpha-neuraminidase, and cathepsin A (protective protein). GALNS copurifies with the complex by different chromatography techniques: affinity chromatography on both cathepsin A-binding and beta-galactosidase-binding columns, gel filtration, and chromatofocusing. Anti-human cathepsin A rabbit antiserum coprecipitates GALNS together with cathepsin A, beta-galactosidase, and alpha-neuraminidase in both a purified preparation of the 1. 27-MDa complex and crude glycoprotein fraction from human placenta extract. Gel filtration analysis of fibroblast extracts of patients deficient in either beta-galactosidase (beta-galactosidosis) or cathepsin A (galactosialidosis), which accumulate KS, demonstrates that the 1.27-MDa complex is disrupted and that GALNS is present only in free homodimeric form. The GALNS activity and cross-reacting material are reduced in the fibroblasts of patients affected with galactosialidosis, indicating that the complex with cathepsin A may protect GALNS in the lysosome. We suggest that the 1.27-MDa complex of lysosomal hydrolases is essential for KS catabolism and that the disruption of this complex may be responsible for the KS accumulation in beta-galactosidosis and galactosialidosis patients.

Animals↗

PubMed1986

Comparison of the enzymatic behavior of high molecular weight and free lysyl-tRNA synthetase from rat liver: kinetic analysis of lysylation of tRNA.

Lysyl-tRNA synthetase occurs in the high molecular weight form in rat liver. The high molecular weight lysyl-tRNA synthetase has been previously demonstrated to exist as multienzyme complexes of aminoacyl-tRNA synthetases. The multienzyme complexes can be dissociated by hydrophobic interaction chromatography and yield fully active, free lysyl-tRNA synthetase. The free form is found to be twice as active as the complexed form in lysylation. Bisubstrate and product inhibition kinetics of lysylation are systematically carried out for highly purified free lysyl-tRNA synthetase and the 18 S synthetase complex. Surprisingly, the two enzyme forms exhibit distinctly different kinetic patterns in bisubstrate and product inhibition kinetics under identical conditions. The 18 S synthetase complex shows kinetic patterns consistent with an ordered bi uni uni bi ping pong mechanism, while the results of free lysyl-tRNA synthetase do not. We conclude that structural organization of lysyl-tRNA synthetase beyond quaternary structure of proteins may alter the enzyme behavior.

Amino Acyl-tRNA Synthetases↗

PubMed1996

The cellulosome: an exocellular, multiprotein complex specialized in cellulose degradation.

Clostridium thermocellum produces a highly active cellulase system that consists of a high-M(r) multienzyme complex termed cellulosome. Hydrolytic components of the cellulosome are organized around a large, noncatalytic glycoprotein termed CipA that acts both as a scaffolding component and a cellulose-binding factor. Catalytic subunits of the cellulosome bear conserved, noncatalytic subdomains, termed dockerin domains, which bind to receptor domains of CipA, termed cohesin domains. CipA includes nine cohesin domains, a cellulose-binding domain, and a specialized dockerin domain. Proteins of the cell envelope carrying cohesin domains that specifically bind the dockerin domain of CipA have been identified. These proteins may mediate anchoring of the cellulosomes to the cell surface. Cellulase complexes similar to the cellulosome of C. thermocellum are produced by several cellulolytic clostridia. High-M(r) multienzyme complexes have also been identified in anaerobic rumen fungi. The architecture of the fungal complexes also seems to rely on the interaction of conserved, noncatalytic docking domains with a scaffolding component. However, the sequence of the fungal docking domains bears no resemblance to the clostridial dockerin domains, suggesting that the fungal and clostridial complexes arose independently.

Bacterial Proteins↗

PubMedDate not supplied

[Supramolecular organization of enzymes of the tricarboxylic acid cycle].

In virtue of analysis of data on the interaction of tricarboxylic acid cycle enzymes with the mitochondrial inner membrane and data on the enzyme-enzyme interactions, the spatial structure for the tricarboxylic acid cycle enzyme complex (tricarboxylic acid cycle metabolon) is proposed. The alpha-ketoglutarate dehydrogenase complex, adsorbed on the mitochondrial inner membrane along one of its 3-fold symmetry axes, plays the key role in the formation of metabolon. Two association sites of the alpha-ketoglutarate dehydrogenase complex located on opposite sides of the complex participate in the interaction with the membrane. The tricarboxylic acid cycle enzyme complex contains one molecule of the alpha-ketoglutarate dehydrogenase complex and six molecules of each of the other enzymes of the tricarboxylic acid cycle, as well as aspartate aminotransferase and nucleosidediphosphate kinase. Succinate dehydrogenase, the integral protein of the mitochondrial inner membrane, is a component of the anchor site responsible for the assembly of metabolon on the membrane. The molecular mass of the complex (ignoring succinate dehydrogenase) is of 8.10(6) daltons. The metabolon symmetry corresponds to the D3 point symmetry group. It is supposed, that the tricarboxylic acid cycle enzyme complex interacts with other multienzyme complexes of the matrix and the electron transfer chain.

Citric Acid Cycle↗

PubMedDate not supplied

[Comparative study of localization of tryptophanyl-tRNA-synthetase and components of high molecular weight aminoacyl-tRNA-synthetase complex in animal cells].

A comparative study on the localization of cytosolic Trp-tRNA synthetase (TrpRS), aminoacyl-tRNA synthetases associated in a multienzyme complex (Glu-tRNA synthetase (GluRS), and Arg-tRNA synthetase (ArgRS)) and polypeptides p37 and p43 from the multienzyme complex was carried out on ultrathin sections of cultured rabbit cells RK-1 by means of immunogold technique. It is shown that GluRS, ArgRS, and polypeptide p43 have approximately the same distribution in the cell as TrpRS. The data obtained evidences in favour of a multienzyme structure of most (or, may be all) aminoacyl-tRNA synthetases in intact cells. A statistical analysis of enzyme distribution in different cell organelles showed nonrandom, compartmentalized distribution of studied synthetases in the mammalian cell. Aminoacyl-tRNA synthetases were found in the cell nucleus in the vicinity of interchromatin granules and in the regions of diffused chromatin. This fact points to a role which these proteins may play in active chromatin functions (transcription, processing, transfer of gene products, etc.) and needs special attention. Detection of ArgRS and GluRS in the nucleus allows one to suggest that either multienzyme synthetase complexes are present not only in the cytoplasm, but also in the nucleus, or these enzymes can dissociate from the complex and pass to the nucleus as individual proteins.

Amino Acyl-tRNA Synthetases↗

PubMed1989

Functional significance of aminoacyl-tRNA synthetase complex in the aminoacylation of tRNA(Leu) isoacceptors.

Aminoacyl-tRNA synthetases, partially purified from rat liver by two different methods, were used in vitro to study aminoacylation profiles of tRNA(Leu) isoacceptors. On the basis of molecular weights, one preparation was similar to the multienzyme complex of aminoacyl-tRNA synthetases, whereas the other apparently represents a partially disrupted complex. In the aminoacylation assay, the multienzyme complex produced a profile of leucyl-tRNA isoacceptors that was similar to those found in vivo and in liver perfusion experiments. The aminoacylation profile that was obtained with the partially disrupted complex varied with the enzyme and leucine concentration used. Especially one of the tRNA(Leu) species was poorly aminoacylated at low leucine and enzyme concentration. These experiments point out that attention should be paid to the nature of the aminoacyl-tRNA synthetase preparation in experiments in which isoacceptor profiles are studied in vitro.

Amino Acyl-tRNA Synthetases↗

PubMed1994

Allosteric interaction of a herpes simplex viral thymidine kinase with host DNA polymerase alpha in mouse LP1-1 cells.

A DNA polymerase alpha-associated multienzyme complex isolated from mouse LP1-1 cells transfected with the thymidine kinase gene of herpes simplex virus type I (1) showed activities of DNA polymerase alpha, topoisomerase II, and thymidine kinase (TK) in the complex. TK antiserum recognized a 43 kDa polypeptide only in the fraction of the multienzyme complex prepared from the LP1-1 cells but not that from L-M(TK-) cells. In permeabilized cells, hydroxyurea did not show any inhibitory effect on either DNA polymerase or TK, whereas aphidicolin, novobiocin, and TK antiserum inhibited both enzymes. These results provide evidence for the functional association and an allosteric interaction between the viral TK and host DNA polymerase alpha.

Allosteric Regulation↗

PubMed1991

Maple syrup urine disease: domain structure, mutations and exon skipping in the dihydrolipoyl transacylase (E2) component of the branched-chain alpha-keto acid dehydrogenase complex.

Maple syrup urine disease (MSUD) is an autosomal recessive disorder in the oxidative decarboxylation of the branched-chain alpha-keto acids derived from leucine, isoleucine and valine. The enzyme deficient in MSUD, the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex, is a mitochondrial multienzyme complex consisting of at least six distinct subunits. MSUD is genetically heterogeneous as manifested by lesions in different subunits of the BCKAD complex among unrelated patients. To approach the biochemical basis of MSUD involving the dihydrolipoyl transacylase (E2) subunit, the domain structure of this polypeptide from human and bovine livers has been defined by limited proteolysis and cDNA cloning. The assembly of 24 E2 subunits into a cubic structure, forming the core of the mammalian BCKAD complex, was established by electron microscopy and sedimentation equilibrium analysis. Highly assembled bovine E2 devoid of prosthetic lipoic acid has been overexpressed in Escherichia coli. Studies carried out with this bacterial expression system have provided insights into the lipoylation process of E2, and the involvement of the His391 residue in the transacylation reaction. At the genetic level, the human E2 gene (DBT) has been regionally assigned to chromosome 1p31, and a related E2 pseudogene to chromosome 3q24 by in situ hybridization. Genomic cloning has shown that the human E2 gene undergoes premature transcriptional termination and alternate splicing as normal events, although its functional significance is unknown. Through the use of the polymerase chain reaction and other recombinant DNA methods, several compound heterozygous mutations at the E2 locus have been identified in classical as well as thiamine-responsive MSUD patients. These mutations would appear to be useful genetic models, which will facilitate investigations into macromolecular organization and protein-protein interactions. Moreover, an array of precise single and multiple exon deletions has been observed in the amplified mutant E2 transcripts. The results represent unexpected secondary effects that are apparently associated with the above primary mutations in the E2 gene.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗