Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Multifunctional Enzymes”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1976

Multifunctional enzyme, bisphosphoglyceromutase/2,3-bisphosphoglycerate phosphatase/phosphoglyceromutase, from human erythrocytes. Evidence for a common active site.

Bisphosphoglyceromutase and 2,3-bisphosphoglycerate phosphatase activities responsible for 2,3-bisphosphoglycerate metabolsim in human red cells are displayed by the same enzyme protein which has phosphoglyceromutase activity [Sasaki, R., et al. (1975) Eur J. Biochem. 50, 581-593]. This enzyme was subjected to chemical modification by trinitrobenzenesulfonate. The three enzyme activities were inactivated by trinitrobenzenesulfonate at the same rate. The sulfhydryl content of the enzyme was unchanged during trinitrophenylation, indicating that derivatization was through the amino group. Trinitrophenylation of about one amino group per mole of the enzyme resulted in complete loss of the three activities. Both 2,3-bisphosphoglycerate and 1,3-bisphosphoglycerate inhibited trinitrophenylation and effectively protected the enzyme from inactivation. Although monophosphoglycerates did not show any protective effect at concentrations which should be adequate based upon their kinetic constants, they were protective at higher concentrations. Inactivation by trinitrophenylation was an apparent first-order reaction. The dissociation constant of the enzyme - 2,3-bisphosphoglycerate complex was determined by analyzing the first-order reaction on the assumption that the protective effect of 2,3-bisphosphoglycerate was due to competition with trinitrobenzenesulfonate. The dissociation constant was in good agreement with kinetic constants of 2,3-bisphosphoglycerate in the enzyme reactions, which indicated that 2,3-bisphosphoglycerate did indeed exert its protective effect through competition with trinitrobenzenesulfonate for an amino group of the enzyme. The protective effect of monophosphoglycerates could be rationalized with kinetic evidence that 2-phosphoglycerate at high concentrations interacts with the 2,3-bisphosphoglycerate binding site. These results indicate that the enzyme exhibits the three enzyme activities at a common active site at which one amino group essential for binding of bisphosphoglycerates is located. Based on the multifunctional properties of this enzyme, a possible mechanism was discussed for regulation of 2,3-bisphosphoglycerate metabolism in human red cells.

Binding Sites↗
PubMed1978

Methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase from porcine liver. Interaction between the dehydrogenase and cyclohydrolase activities of the multifunctional enzyme.

Methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.5) one of the activities of a trifunctional folate-dependent enzyme from porcine liver, uses an ordered kinetic mechannism as determined from initial velocity, product inhibition and dead-end inhibition studies. The final product released from the dehydrogenase is methenyltetrahydrofolate. However, from the time course of appearance of products it is observed that the methenyltetrahydrofolate, rather than equilibrating with the solution, is converted preferentially to formyltetrahydrofolate by the cyclohydrolase, (5,10-methenyletrahydrofolate 5-hydrolase (decyclizing), EC 3.5.4.9) demonstrating a functional interaction between these two enzymic activites.

Aminohydrolases↗
PubMed1977

phiX174 cistron A protein is a multifunctional enzyme in DNA replication.

The cistron A protein induced by phage varphiX174 nicks (produces a single-strand break in) the viral strand of the superhelical varphiX duplex DNA, thereby forming a complex with the DNA. The protein, seen bound to the DNA in the electron microscope, was located in the restriction endonuclease fragment between nucleotides 4290 and 4330 on the varphiX map [Sanger, F., Air, G. M., Barrel, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocomb, P. M. Y. & Smith, M. (1977) Nature 265, 687-695]. Replication also was initiated at this point, thus identifying the site of cistron A protein nicking and binding as the origin of replication. The cisA-DNA complex (separated from free cistron A protein), upon the addition of Escherichia coli rep protein, ATP, and DNA binding protein, is unwound to generate a single-stranded linear [presumably the nicked (+) strand] and a circular [presumably the (-) strand] molecule. The cisA-DNA complex, upon the further addition of DNA polymerase III holoenzyme and deoxynucleoside triphosphates, supports replication to generate viral, single-stranded circles, as many as 15 circles per cisA-DNA complex. The replicating intermediates seen in the electron microscope are a novel form of "rolling circle" [Gilbert, W. & Dressler, D. H. (1969) Cold Spring Harbor Symp. Quant. Biol. 33, 473-485]. The 5' end (presumably with the cistron A protein bound to it) is locked in the replication fork and loops back to accompany the strand-separation and replication fork around the template [(-) strand] circle. Thus, the multiple functions of cistron A protein include: (i) nicking the viral strand at the origin of replication to initiate a round of replication, (ii) participating in a complex which supports fork movement in strand separation and replication, (iii) nicking again at the regenerated origin to produce a unit-length DNA, and (iv) ligating the newly generated 3'-OH end to the 5'-phosphate-complexed end to form a circular viral molecule.

Coliphages↗
PubMed1979

Characterization of pyrimidine deoxyribonucleoside kinase (thymidine kinase) and thymidylate kinase as a multifunctional enzyme in cells transformed by herpes simplex virus type 1 and in cells infected with mutant strains of herpes simplex virus.

Pyrimidine deoxyribonucleoside kinase (thymidine kinase [TK]) was purified from two herpes simplex virus type 1 (HVS-1)-transformed TK-deficient mouse (LMTK-) cell lines and from LMTK- cells infected with HSV-1 mutant viruses coding for variant TK enzymes. These preparations exhibited normal or variant virus-induced thymidylate kinase activities correlating with their relative TK activities. Neither virus-induced activity was detected in LMTK- cells infected with an HSV-1 TK-deficient mutant. These results suggest that HSV-1 thymidylate kinase activity and TK activity are mediated by the same protein.

Animals↗
PubMed1979

Purification and characterization of phiX174 gene A protein. A multifunctional enzyme of duplex DNA replication.

Synthesis of phiX174 viral (+) strand circles in vitro requires gene A protein, rep protein, DNA binding protein, and DNA polymerase III holoenzyme (Eisenberg, S., Scott, J. F., and Kornberg, A., (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3151-3155). We have used this reaction as an assay to isolate gene A protein in greater than 90% purity. Its molecular weight under denaturing conditions is 59,000. The protein tends to aggregate and lose activity at low ionic strength. Tritium-labeled gene A protein cleaves the phiX174 duplex replicative form and is bound to it in a 1:1 ratio as part of an active replication complex. The attachment, at the 5' phosphoryl end of the cleavage point, is apparently covalent. The complex was not dissociated by: (i) banding in CsCl, (ii) treatment with 0.2 M NaOH, or (iii) boiling in 1% sodium dodecyl sulfate and electrophoresis on a sodium dodecyl sulfate-acrylamide gel; only micrococcal nuclease digestion of the DNA released the protein.

Coliphages↗
PubMed1979

Effect of estrogen on fatty acid synthetase in the chicken oviduct and liver.

Estrogen administered to one-month-old female chickens resulted in a 180-fold increase in the amount of fatty acid synthetase, a seven-fold increase in the enzyme activity per gram of tissue and a 25-fold increase in the weight of the oviduct. In contrast, the fatty acid synthetase content in liver increased three-fold; activity per gram of tissue increased two-fold and the weight increased two-fold. The large increase in the fatty acid synthetase activity in the oviduct was due to a corresponding increase in the amount of the fatty acid synthetase protein since the specific activities of highly purified preparations of oviduct and liver fatty acid synthetases were the same and the two enzymes had the same end point as determined by immunoprecipitation. That the increase in activity of the oviduct enzyme is not due to a modification was further supported by physicochemical comparison of the oviduct enzyme with the chicken liver enzyme. Thus, the synthetase complexes have similar size, their subunit composition and size appear to be the same, and both are multifunctional enzymes. Finally, kinetic studies and product analyses indicated no catalytic difference between the enzyme induced by estrogen in the oviduct and the liver enzyme.

Adipose Tissue↗
PubMed1977

Pyrimidine biosynthesis in Serratia marcescens: polypeptide interactions of three nonsequential enzymes.

Orotidine-5'-monophosphate pyrophosphorylase (OMPppase, E.C. 2.4.2.10) and orotidylate decarboxylase (OMPdecase, E.C. 4.1.1.23) were purified from Serratia marcescens HY. These enzymes required physical association for maximal catalytic activities and formed a fragile complex with dihydroorotase (DHOase, E.C. 3.5.2.3). OMPppase reversibly lost 50% of its activity upon separation from DHOase. The kinetic characteristics of OMPppase were modified by this separation. In the presence of DHOase, the Kms for PRPP and orotate were stoichiometric: 2.3 X 10(-6) M and 2.6 X 10(-6) M, respectively. Following separation, the Kms were significantly different: 1.3 X 10(-6) M for PRPP and 4.1 X 10(-6) M for orotate. OMPppase and OMPdecase could be reversibly separated by acrylamide gel electrophoresis, but the separation was accompanied by a loss of catalytic efficiency for both enzymes. DHOase readily associated into multiple molecular forms and could not be purified. The DHOase-OMPppase-OMPdecase interactions demonstrate that a weakly aggregated, multifunctional enzyme complex participates in the biosynthesis of pyrimidine nucleotides in S. marcescens. This unique association of non-sequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.

Carboxy-Lyases↗
PubMed1979

Characterization of thymidine kinase and phosphorylation of deoxyribonucleosides in Chlamydomonas reinhardti.

Using gel filtration chromatography, we find a single peak of deoxythymidine phosphorylating activity in Chlamydomonas reinhardti. This activity has characteristics of a thymidine kinase, in that (1) it will utilize ATP (or dATP) or CTP (or dCTP) as phosphoryl donor, but not AMP or phenyl phosphate, and (2) it is inhibited by dTTP (and less so by dTDP, dUTP, and dUDP) but is unaffected by 3'-5' cyclic AMP. Partially purified chlamydomonas thymidine kinase has a pH optimum near 8.5, and a molecular weight of 80,000 to 85,000 daltons. Kinetic studies indicate a ping-pong mechanism with a Km for thymidine of 1.5 x 10(-7) moles per liter. 5-Bromo- and 5-fluorodeoxyuridine, and to a lesser degree deoxyuridine, are competitive inhibitors, but significant phosphorylation of these nucleotides could not be demonstrated in vitro by thymidine kinase. While thymidine is phosphorylated to dTMP by crude Chlamydomonas extracts, greater than 80% of the product formed by the partially purified enzyme is dTTP. Further, the gel filtration elution position of the single deoxythymidylate kinase activity present in cell extracts coincides with that of thymidine kinase. These results suggest that a multifunctional enzyme, rather than three separate phosphorylating activities, may be responsible for dTTP formation.

Adenosine Triphosphate↗
PubMed2026

Functional identification of the key gene Eh-fadB in nicosulfuron degradation by Enterobacter hormaechei ES1 based on multi-omics and enzymatic characterization.

Nicosulfuron is a sulfonylurea herbicide with residues that pose ecological risks in agricultural soils. Here we elucidated the degradation mechanism of Enterobacter hormaechei ES1 through whole-genome sequencing, transcriptomics, metabolomics, gene knockout, heterologous expression, and soil bioremediation assays. Under nicosulfuron stress, ES1 upregulated antioxidant enzymes including SOD, POD, and CAT, along with glutathione synthesis, to scavenge excess reactive oxygen species. HPLC-TOF-MS identified degradation intermediates such as ADMP and ASDM, indicating initial cleavage of the sulfonylurea bridge. Integrated multi-omics prioritized Eh-fadB, encoding a fatty acid β-oxidation multifunctional enzyme, as a novel degradative gene. Targeted knockout of Eh-fadB reduced nicosulfuron degradation from 87.6% to 37.04%, while genetic complementation restored nearly full activity. Purified Eh-FadB directly converted nicosulfuron, with optimal performance at 30 °C and pH 5-6; its activity was enhanced by Na+ and Pb2+ but inhibited by Fe3+. Molecular docking and dynamics identified His-450 and Asn-427 as key residues for substrate binding. In contaminated soil, inoculation with ES1 reduced nicosulfuron content within 21 days and promoted recovery of dehydrogenase and urease activities. This study provides the first genetic and biochemical evidence that a FadB-type enzyme participates in nicosulfuron catabolism, supporting sulfonylurea bridge cleavage and its potential for soil bioremediation.

Eh-fadB↗
PubMed1976

Microsomal membrane permeability and the hepatic glucose-6-phosphatase system. Interactions of the system with D-mannose 6-phosphate and D-mannose.

We have proposed that glucose-6-phosphatase (EC 3.1.3.9) is a two-component system consisting of (a) a glucose-6-P-specific transporter which mediates the movement of the hexose phosphate from the cytosol to the lumen of the endoplasmic reticulum (or cisternae of the isolated microsomal vesicle), and (b) a nonspecific phosphohydrolase-phosphotransferase localized on the luminal surface of the membrane (Arion, W.J., Wallin, B.K., Lange, A.J., and Ballas, L.M. (1975) Mol. Cell. Biochem. 6, 75-83). Additional support for this model has been obtained by studying the interactions of D-mannose-6-P and D-mannose with the enzyme of untreated (i.e. intact) and taurocholate-disrupted microsomes. An exact correspondence was shown between the mannose-6-P phosphohydrolase activity at low substrate concentrations and the permeability of the microsomal membrane to EDTA. The state of intactness of the membrane influenced the kinetics of mannose inhibition of glucose-6-P hydrolysis; uncompetitive and noncompetitive inhibitions were observed for intact and disrupted microsomes, respectively. The apparent Km for glucose-6-P was smaller with intact preparations at mannose concentrations above 0.3 M. Mannose significantly inhibited total glucose-6-P utilization by intact microsomes, whereas D-glucose had a stimulatory effect. Both hexoses markedly enhanced the rate of glucose-6-P utilization by disrupted microsomes. The actions of mannose on the glucose-6-phosphatase of intact microsomes fully support the postulated transport model. They are predictable consequences of the synthesis and accumulation of mannose-6-P in the cisternae of microsomal vesicles which possess a nonspecific, multifunctional enzyme on the inner surface and a limiting membrane permeable to D-glucose, D-mannose, glucose-6-P, but impermeable to mannose-6-P. The latency of the mannose-6-P phosphohydrolase activity is proposed as a reliable, quantitative index of microsomal membrane integrity. The inherent limitations of the use of EDTA permeability for this purpose are discussed.

Animals↗
PubMed1976

Fine structure of the URA2 locus in Saccharomyces cerevisiae. II. Meiotic and mitotic mapping studies.

The URA2 locus codes for a multifunctional enzyme complex carrying aspartate transcarbamylase (ATCase) and carbamyly phosphate synthetase (CPSase) activities. Three different types of ura2 mutants were tested in meiotic and mitotic recombination experiments: ura2A mutants devoid of ATCase activity, ura2C mutants devoid of CPSase activity and ura2B mutants devoid of both activities. All the ura2A mutations were found to be clustered at one end of the URA2 locus, called zone A, while the ura2C mutations were localized in a region at the other end, called zone C. All but two ura2B mutations (most of them suppressible) were distributed throughout zone C; the two ura2B exceptions which are small deletions, mapped in zone A. On the meiotic as well as on the mitotic map an intermediary or dead-space zone is located between zones A and C. No mutation has yet been found to map in this zone. The relative lengths of the three zones A, intermediary and C are 1 :2-3 :3-4, respectively. These data are consistent with the hypothesis that the URA2 locus consisting of at least two cistrons: C (CPSase) and A (ATCase), is transcribed into a single polycistronic message in the direction C to A. However, alternative hypotheses in reference to Peterson and MacLaughlin's observations (1973) are discussed.

Aspartate Carbamoyltransferase↗
PubMed2026

Functional analysis of the role of a wound-induced leucine aminopeptidase gene homologue isolated from Rorippa indica in aphid herbivory.

Leucine aminopeptidases (LAPs) are multifunctional enzymes with roles in both defence and development. In plants, they are reported to be induced by wound-inflicting Lepidopteran insects and regulate wound response pathways leading to an effective defence response. Infestation by Hemipteran mustard aphid, Lipaphis erysimi (L.) Kaltenbach has been reported to induce wound response as well as a wound-responsive Arabidopsis thaliana Lap1 homologue (RI01; GenBank Accession: JK034053) in Rorippa indica (L.) Hiern. This is interesting as Hemipteran insects like aphids are assumed to inflict minimal wounding. In the present study, starting with the RI01 sequence information, we isolated the full length (1566 bp) sequence of a novel R. indica Lap (RiLap) gene, performed in silico analyses and developed transgenic R. indica plants with suppressed RiLAP activity by expressing a 565 bp antisense fragment of RiLap cDNA. We found that the isolated RiLAP is an acidic LAP of M17 family and suppressing it causes a significant increase in aphid herbivory but reduction in total chlorophyll content and possibly photosynthetic capacity in aphid infested transgenic plants of the T1 generation. These findings though preliminary suggest that RiLap could have a role in deterring aphids by acting as a regulatory protein simultaneously balancing defence response and photosynthetic capacity or plant growth. Noting the dearth of research in this area, this pilot study will be useful for designing future in depth analyses in understanding the role of Laps in defence response against Hemipteran insects. The study has implications in the development of sustainable pest management avenues.

Leucyl Aminopeptidase↗
PubMed1978

Association of thymidylate kinase activity with pyrimidine deoxyribonucleoside kinase induced by herpes simplex virus.

Thymidine kinase derived from LMTK+ does not exhibit thymidylate kinase activity. However, protein isolated by affinity column chromatography from thymidine kinase-deficient mouse cells (LMTK-) infected by herpes simplex virus type 1 shows thymidylate kinase activity in addition to thymidine kinase and deoxycytidine kinase activities. The virus-induced multifunctional enzyme has a molecular weight of 85,000, whereas the molecular weight of thymidylate kinase from uninfected LMTK- mouse cells is 71,000. The virus-induced enzyme has a Km for thymidine of 0.8 micromolar, and for thymidylate of 25 micromolar, and for thymidylate of 25 micromolar; the ratio of Vmax for thymidylate kinase to thymidine kinase is 1.7. When subjected to isoelectric focusing, thymidylate kinase activity is not separated from thymidine kinase activity, and even though four peaks of activity are observed they have a constant ratio of thymidylate kinase to thymidine kinase activity. The isoelectric points (pI) of these four peaks are 4.8, 5.8, 6.2, and 6.6, respectively. Thymidylate kinase, derived from uninfected cells when subjected to isoelectric focusing, separates into a major component with an isoelectric point at pH 8.2 and a minor component at pH 7.7. Although thymidine and thymidylate kinase activities derived from the virus-infected cells cannot be separated either by affinity column chromatography, glycerol density gradient centrifugation, or isoelectric focusing, there is a differential rate of inactivation when the enzyme is subjected to incubation at 37 degrees, with thymidylate kinase activity being more labile than thymidine kinase activity.

Enzyme Induction↗
PubMed1979

[Monkey liver microsomal glucose-6-phosphatase].

Kinetic studies indicate that glucose-6-phosphatase is a multifunctional enzyme. a) Phosphohydrolase activities. The mannose-6-phosphatase activity is low (Km = 8 mM, VM = 90 nmoles. min-1mg-1). The enzyme shows a strong affinity for glucose-6-phosphate (Km = 2.5 mM, VM = 220 nmoles.min-1mg-1). beta-glycerophosphate (K1 = 30 mM), D-glucose (Ki = 120 mM) are mixed type inhibitors; pyrophosphate (Ki = 2 mM) is a non competitive one. b) Phosphotransferase activities. Di and triphosphate adenylic nucleosides or phosphoenol pyruvate are not substrates. Carbamylphosphate serves as a phosphoryl donor with D-glucose as acceptor. The phosphate transfer is consisstent with a random mechanism in which the binding of one substrate increases the enzymes affinity for the second substrate. Apparent Km values for carbamyl-phosphate range from 5.2 mM (D-glucose concentration leads to infinity) to 8 mM (D-glucose concentration leads to 0). The corresponding apparent Km values for D-glucose are 59 mM (carbamyl-phosphate concentration leads to infinity) to 119 mM (carbamyl-phosphate concentration leads to 0). Maximal reaction velocity with infinite levels of both substrates is 270 nmoles.min-1.mg-1. Pyrophosphate is a poor phosphoryl donnor (Km = 55 mM with D-glucose concentration 250 mM). In addition we do not find any latency; detergents, namely sodium deoxycholate, Triton X 100 do not affect or inhibit glucose-6-phosphatase activity.

Animals↗