PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “MUTASES”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 1,297 records · Page 72Linked to original sources
PubMed2003

The first experimental test of the hypothesis that enzymes have evolved to enhance hydrogen tunneling.

The literature hypothesis that "the optimization of enzyme catalysis may entail the evolutionary implementation of chemical strategies that increase the probability of quantum-mechanical tunneling" is experimentally tested herein for the first time. The system employed is the key to being able to provide this first experimental test of the "enhanced hydrogen tunneling" hypothesis, one that requires a comparison of the three criteria diagnostic of tunneling (vide infra) for the same, or nearly the same, reaction with and without the enzyme. Specifically, studied herein are the adenosylcobalamin (AdoCbl, also known as coenzyme B(12))-dependent diol dehydratase model reactions of (i). H(D)(*) atom abstraction from ethylene glycol-d(0) and ethylene glycol-d(4) solvent by 5'-deoxyadenosyl radical (Ado(*)) and (ii.) the same H(*) abstraction reactions by the 8-methoxy-5'-deoxyadenosyl radical (8-MeOAdo(*)). The Ado(*) and 8-MeOAdo(*) radicals are generated by Co-C thermolysis of their respective precursors, AdoCbl and 8-MeOAdoCbl. Deuterium kinetic isotope effects (KIEs) of the H(*)(D(*)) abstraction reactions from ethylene glycol have been measured over a temperature range of 80-120 degrees C: KIE = 12.4 +/- 1.1 at 80 degrees C for Ado(*) and KIE = 12.5 +/- 0.9 at 80 degrees C for 8-MeOAdo(*) (values ca. 2-fold that of the predicted maximum primary times secondary ground-state zero-point energy (GS-ZPE) KIE of 6.4 at 80 degrees C). From the temperature dependence of the KIEs, zero-point activation energy differences ([E(D) - E(H)]) of 3.0 +/- 0.3 kcal mol(-)(1) for Ado(*) and 2.1 +/- 0.6 kcal mol(-)(1) for 8-MeOAdo(*) have been obtained, both of which are significantly larger than the nontunneling, zero-point energy only maximum of 1.2 kcal mol(-)(1). Pre-exponential factor ratios (A(H)/A(D)) of 0.16 +/- 0.07 for Ado(*) and 0.5 +/- 0.4 for 8-MeOAdo(*) are observed, both of which are significantly less than the 0.7 minimum for nontunneling behavior. The data provide strong evidence for the expected quantum mechanical tunneling in the Ado(*) and 8-MeOAdo(*)-mediated H(*) abstraction reactions from ethylene glycol. More importantly, a comparison of these enzyme-free tunneling data to the same KIE, (E(D) - E(H)) and A(H)/A(D) data for a closely related, Ado(*)-mediated H(*) abstraction reaction from a primary CH(3)- group in AdoCbl-dependent methylmalonyl-CoA mutase shows the enzymic and enzyme-free data sets are identical within experimental error. The Occam's Razor conclusion is that at least this adenosylcobalamin-dependent enzyme has not evolved to enhance quantum mechanical tunneling, at least within the present error bars. Instead, this B(12)-dependent enzyme simply exploits the identical level of quantum mechanical tunneling that is available in the enzyme-free, solution-based H(*) abstraction reaction. The results also require a similar, if not identical, barrier width and height within experimental error for the H(*) abstraction both within, and outside of, the enzyme.

Cobamides↗
PubMed2005

How the Co-C bond is cleaved in coenzyme B12 enzymes: a theoretical study.

The homolytic cleavage of the organometallic Co-C bond in vitamin B12-dependent enzymes is accelerated by a factor of approximately 10(12) in the protein compared to that of the isolated cofactor in aqueous solution. To understand this much debated effect, we have studied the Co-C bond cleavage in the enzyme glutamate mutase with combined quantum and molecular mechanics methods. We show that the calculated bond dissociation energy (BDE) of the Co-C bond in adenosyl cobalamin is reduced by 135 kJ/mol in the enzyme. This catalytic effect can be divided into four terms. First, the adenosine radical is kept within 4.2 angstroms of the Co ion in the enzyme, which decreases the BDE by 20 kJ/mol. Second, the surrounding enzyme stabilizes the dissociated state by 42 kJ/mol using electrostatic and van der Waals interactions. Third, the protein itself is stabilized by 11 kJ/mol in the dissociated state. Finally, the coenzyme is geometrically distorted by the protein, and this distortion is 61 kJ/mol larger in the Co(III) state. This deformation of the coenzyme is caused mainly by steric interactions, and it is especially the ribose moiety and the Co-C5'-C4' angle that are distorted. Without the polar ribose group, the catalytic effect is much smaller, e.g. only 42 kJ/mol for methyl cobalamin. The deformation of the coenzyme is caused mainly by the substrate, a side chain of the coenzyme itself, and a few residues around the adenosine part of the coenzyme.

Carbon↗
PubMed2005

Isotope effects on the enzymatic and nonenzymatic reactions of chorismate.

The important biosynthetic intermediate chorismate reacts thermally by two competitive pathways, one leading to 4-hydroxybenzoate via elimination of the enolpyruvyl side chain, and the other to prephenate by a facile Claisen rearrangement. Measurements with isotopically labeled chorismate derivatives indicate that both are concerted sigmatropic processes, controlled by the orientation of the enolpyruvyl group. In the elimination reaction of [4-2H]chorismate, roughly 60% of the label was found in pyruvate after 3 h at 60 degrees C. Moreover, a 1.846 +/- 0.057 2H isotope effect for the transferred hydrogen atom and a 1.0374 +/- 0.0005 18O isotope effect for the ether oxygen show that the transition state for this process is highly asymmetric, with hydrogen atom transfer from C4 to C9 significantly less advanced than C-O bond cleavage. In the competing Claisen rearrangement, a very large 18O isotope effect at the bond-breaking position (1.0482 +/- 0.0005) and a smaller 13C isotope effect at the bond-making position (1.0118 +/- 0.0004) were determined. Isotope effects of similar magnitude characterized the transformations catalyzed by evolutionarily unrelated chorismate mutases from Escherichia coli and Bacillus subtilis. The enzymatic reactions, like their solution counterpart, are thus concerted [3,3]-sigmatropic processes in which C-C bond formation lags behind C-O bond cleavage. However, as substantially larger 18O and smaller 13C isotope effects were observed for a mutant enzyme in which chemistry is fully rate determining, the ionic active site may favor a somewhat more polarized transition state than that seen in solution.

Chorismic Acid↗
PubMed2005

Theoretical studies of dissociative phosphoryl transfer in interconversion of phosphoenolpyruvate to phosphonopyruvate: solvent effects, thio effects, and implications for enzymatic reactions.

The conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr) is catalyzed by PEP mutase via a dissociative mechanism. In this work, we investigate the uncatalyzed reaction using ab initio methods, density functional theory, and the semiempirical MNDO/d method. Comparisons of geometries and relative energies of stationary points (minima and transition states) with density functional results indicate that the semiempirical method is reasonably accurate. Solvent effects are examined using implicit solvent models, including the recently extended smooth conductor-like screening model. Due to the large negative charge carried by the system, solvation is found to drastically alter the location and energy of stationary points along the dissociative reaction pathways. The influence of substituting a nonbridging phosphoryl oxygen by sulfur (thio effects) was also investigated. Implications of these results for the enzymatic reaction are discussed.

Calibration↗
PubMedDate not supplied

Proteomic characterization of rat liver exposed to 2,3,7,8-tetrachlorobenzo-p-dioxin.

Dioxins are a class of polyhalogenated aromatic hydrocarbons that induce a wide spectrum of toxic responses in experimental animals. In this study, 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) was exposed to two SD rat groups; one group for short-term exposure at a single dose of 1, 10, 20 and 50 mug/kg body weight (group 1) and the other for long-term exposure at daily and-low dose of 0.01, 0.1, 1 and 2.5 microg/kg body weight (group 2) for a month. Two-dimensional electrophoresis (2-DE) was utilized to resolve the protein profile of rat liver exposed to TCDD at different doses. In the analysis of 2-DE of the group 1, two new-expressed spots and seven volume-increased spots were detected and identified by ESI-Q-TOF MS/MS; especially, proteasome subunit beta type 3 was increased in all doses. In addition, in the group 2, six volume-increased spots were screened; particularly, histidine triad nucleotide binding protein was increased in both 0.1 microg/kg dose and 1 microg/kg dose. The identified proteins were confirmed using Western blot. Among the identified proteins, apolipoprotein A-IV may protect lipid peroxidation and atherosclerosis induced by TCDD exposure and the expression level of phosphoglycerate mutase increases due to hyperthyroidism induced by TCDD exposure.

Amino Acid Sequence↗
PubMed2005

Monochloroacetic acid inhibits liver gluconeogenesis by inactivating glyceraldehyde-3-phosphate dehydrogenase.

We previously reported that a lethal dose of monochloroacetate (MCA) causes severe hypoglycemia and lactic acidosis. MCA has been thought to inhibit mitochondrial aconitase; however, the exact effect of MCA on hepatic glucose metabolism is not clear. In this study, we investigated the effects of MCA on liver gluconeogenesis using an isolated perfused rat liver system. Gluconeogenesis from 2.5 mM lactate was inhibited by 1 mM MCA and was completely abolished after 2 h of perfusion. Levels of citric acid cycle intermediates such as citrate, isocitrate, and 2-oxoglutarate (2-OG) were significantly reduced by MCA. The finding that the levels of citrate and 2-OG were similarly reduced (to 31 and 36% of control, respectively) indicates that aconitase was not inhibited by MCA. On the contrary, gluconeogenesis from glycerol, which can be converted to glucose without glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was not inhibited by MCA. GAPDH was inactivated by MCA in vitro, but enolase, phosphoglycerate mutase, and phosphoglycerate kinase were not inactivated at the same or higher concentrations of MCA. Furthermore, GAPDH activity in the MCA-perfused liver decreased to 33-42% of control and that in the liver of rats exposed to MCA was reduced to 19% of control. We concluded that MCA inactivates GAPDH, and this is the cause of the inhibition of liver gluconeogenesis.

Acetates↗
PubMed1997

Clinical heterogeneity and prognosis in combined methylmalonic aciduria and homocystinuria (cblC).

The clbC form of methylmalonic acidaemia is a rare and poorly understood condition which results from impaired biosynthesis of methylcobalamin and adenosylcobalamin. The consequent functional deficiencies of methylmalonyl-CoA mutase and methionine synthase produce both methylmalonic aciduria and homocystinuria. Systemic symptoms and neurological decompensation comprise the clinical phenotype. In an effort to clarify the phenotype and prognosis, we obtained clinical information on 50 patients with methylmalonic acidaemia whose cells had been assigned to the cblC complementation group. We identified two distinct phenotypes; they differed in age of onset, presence of systemic symptoms, type of neurological symptoms, and outcome after diagnosis and treatment. Forty-four patients presented in the first year of life. Feeding difficulties, neurological dysfunction (hypotonia, seizures, developmental delay), and ophthalmological and haematological abnormalities characterized their clinical picture. About one-quarter of those patients died. Survival was associated with neurological impairment; only one infant was neurologically intact at follow-up. Onset in childhood, in contrast, was associated with less severe haematological abnormalities, largely involving the red cell series. Extrapyramidal signs, dementia, delirium or psychosis characterized the neurological findings. Survival, with mild to moderate disability in some, was typical in patients with later onset. Treatment in both groups included hydroxycobalamin, betaine and carnitine; complete normalization of biochemical parameters was rare.

Adolescent↗
PubMed1999

Progressive neurological deterioration and MRI changes in cblC methylmalonic acidaemia treated with hydroxocobalamin.

Cobalamin C (cblC) defects result in decreased activity of both methylmalonyl-CoA mutase and N5-methyltetrahydrofolate:homocysteine methyltransferase (methionine synthase), with subsequent methylmalonic acid-uria and homocystinuria. Patients typically show failure to thrive, developmental delay and megaloblastic anaemia. Vitamin B12 therapy has been beneficial in some cases. We report a now 4-year-old Hispanic girl with cblC disease documented by complementation analysis, with progressive neurological deterioration and worsening head MRI changes while on intramuscular hydroxocobalamin begun at age 3 weeks. Oral carnitine and folic acid were added at age 1 year. Blood levels of methylmalonic acid were reduced to treatment ranges. In the absence of acute metabolic crises, she developed microcephaly, progressive hypotonia and decreased interactiveness. Funduscopic examination was normal at age 13 months. At age 19 months, she developed nystagmus, and darkly pigmented fundi and sclerotic retinal vessels were observed on examination. Her neonatal head MRI was normal. By age 1 year, the MRI showed diffuse white-matter loss with secondary third and lateral ventricle enlargement, a thin corpus callosum, and normal basal ganglia. At age 15 months, progression of the white-matter loss, as well as hyperintense globi pallidi, were present. Interval progression of both grey- and white-matter loss was seen at age 27 months. We therefore caution that progressive neurological deterioration and head MRI abnormalities may still occur in cblC disease, despite early initiation of hydroxocobalamin therapy and improvement in toxic metabolite concentrations in physiological fluids.

Brain↗
PubMed2001

Mapping the ligand binding site at protein side-chains in protein-ligand complexes through NOE difference spectroscopy.

This report describes a novel NMR approach for mapping the interaction surface between an unlabeled ligand and a 13C,15N-labeled protein. The method relies on the spin inversion properties of the dipolar relaxation pathways and records the differential relaxation of two spin modes, where ligand and protein 1H magnetizations are aligned either in a parallel or anti-parallel manner. Selective inversion of protein protons is achieved in a straightforward manner by exploiting the one-bond heteronuclear scalar couplings (1J(CH), 1J(NH)). Suppression of indirect relaxation pathways mediated by bulk water or rapidly exchanging protons is achieved by selective inversion of the water signal in the middle of the NOESY mixing period. The method does not require deuteration of the protein or well separated spectral regions for the protein and the ligand, respectively. Additionally, in contrast to previous methods, the new experiment identifies side-chain enzyme ligand interactions along the intermolecular binding interface. The method is demonstrated with an application to the B12-binding subunit of glutamate mutase from Clostridium tetanomorphum for which NMR chemical shift changes upon B12-nucleotide loop binding and a high-resolution solution structure are available.

Bacterial Proteins↗
PubMed2001

Simultaneous measurement of intra- and intermolecular NOEs in differentially labeled protein-ligand complexes.

A new NOE strategy is presented that allows the simultaneous observation of intermolecular and intramolecular NOEs between an unlabeled ligand and a 13C,15N-labeled protein. The method uses an adiabatic 13C inversion pulse optimized to an empirically observed relationship between 1 J(CH) and carbon chemical shift to selectively invert the protein protons (attached to 13C). Two NOESY data sets are recorded where the intermolecular and intramolecular NOESY cross peaks have either equal or opposite signs, respectively. Addition and subtraction yield two NOESY spectra which contain either NOEs within the labeled protein (or unlabeled ligand) or along the binding interface. The method is demonstrated with an application to the B12-binding subunit of Glutamate Mutase from Clostridium tetanomorphum complexed with the B12-nucleotide loop moiety of the natural cofactor adenosylcobalamin (Coenzyme B12).

Binding Sites↗
PubMed2001

Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus.

Flocculation of yeasts is a cell-cell aggregation phenomenon which is driven by interactions between cell wall lectins and cell wall heteropolysaccharides. In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent. Incubation of flocculent K. bulgaricus cells with EDTA or Hecameg led to extracts showing hemagglutinating and flocculating properties. Purification of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K. bulgaricus. Both bands with specific reflocculating activity were composed of five subunits, of which only three possessed weak reflocculating activity upon deflocculated yeast. The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex. These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively. However, no such enzymatic activity could be detected in the crude extract issued from treatment with EDTA and Hecameg of flocculent yeast cells. When yeasts had grown in glucose poor medium, flocculation was drastically affected. The EDTA and Hecameg crude extracts showed weak reflocculating activity. After PAGE, the protein complexes did not appear in the EDTA extract, but they did appear in the Hecameg crude extract. These results suggest that: (i) self-flocculation of K. bulgaricus depends on the expression of different floc-forming protein complex, (ii) these proteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.

Amino Acid Sequence↗
PubMed2003

Backbone resonance assignment in large protonated proteins using a combination of new 3D TROSY-HN(CA)HA, 4D TROSY-HACANH and 13C-detected HACACO experiments.

A new method for backbone resonance assignment suitable for large proteins with the natural (1)H isotope content is proposed based on a combination of the most sensitive TROSY-type triple-resonance experiments. These techniques include TROSY-HNCO, (13)C'-detected 3D multiple-quantum HACACO and the newly developed 3D TROSY multiple-quantum-HN(CA)HA and 4D TROSY multiple-quantum-HACANH experiments. The favorable relaxation properties of the multiple-quantum coherences, signal detection using the (13)C' antiphase coherences, and the use of TROSY optimize the performance of the proposed set of experiments for application to large protonated proteins. The method is demonstrated with the 44 kDa uniformly (15)N,(13)C-labeled and fractionally (35%) deuterated trimeric B. Subtilis Chorismate Mutase and is suitable for proteins with large correlation times but a relatively small number of residues, such as membrane proteins embedded in micelles or oligomeric proteins.

Carbon Isotopes↗
PubMed1980

Levels of 2,3-diphosphoglycerate in Friend leukaemic cells.

Most cells are thought to contain trace amounts of 2,3-diphosphoglycerate (DPG), as it acts as a cofactor in the interconversion of 2-phosphoglycerate and 3-phosphoglycerate by the glycolytic enzyme phosphoglyceromutase. DPG is synthesized from 1,3-diphosphoglycerate by the action of diphosphoglycerate mutase. Lowry et al. reported levels of 29 mumol DPG per kg wet weight brain tissue which is approximately 3 pmol per 10(8) cells, assuming that 1 g of brain tissue contains 10(9) cells. In contrast, erythroid cells contain 50-100 nmol DPG per 10(8) cells, depending on the species and the stage of development. This is of the order of a 1,000-fold more DPG compared with non-erythroid cells. In red cells DPG concentration modulates the binding of oxygen to haemoglobin. I show here that erythroid precurser cells also contain markedly raised levels of DPG.

Animals↗
PubMed1988

Phosphonate biosynthesis: isolation of the enzyme responsible for the formation of a carbon-phosphorus bond.

The first isolation of a naturally occurring phosphonate in 1959 led rapidly to the discovery of a variety of metabolites containing a phosphorus-carbon bond. Phosphonates have been found in bacteria, fungi, and higher organisms such as the snail schistosome vector Biomphalaria. The biosynthetic path to the P-C bond has, however, remained undefined. Thus although it was shown twenty years ago that the isotope label from [14C]glucose or from [32P]phosphoenolpyruvate is incorporated into 2-aminoethylphosphonate by the protozoan Tetrahymena pyriformis, the presumed stoichiometric transformation of phosphoenolpyruvate to phosphonopyruvate has never been demonstrated. Low conversions of phosphoenolpyruvate into 2-aminoethylphosphonate and the trapping of phosphonopyruvate from phosphoenolpyruvate have been reported, but these reactions have not proved reproducible, and the existence of the critical enzyme, phosphoenolpyruvate phosphonomutase, has remained notional. We now report experiments that resolve this enigma, and describe the isolation and characterization of the pure mutase from T. pyriformis.

Amino Acid Sequence↗
PubMed1997

The interplay between binding energy and catalysis in the evolution of a catalytic antibody.

Antibody catalysis provides an opportunity to examine the evolution of binding energy and its relation to catalytic function in a system that has many parallels with natural enzymes. Here we report such a study involving an antibody AZ-28 that catalyses an oxy-Cope rearrangement, a pericyclic reaction that belongs to a well studied and widely used class of reactions in organic chemistry. Immunization with transition state analogue 1 results in a germline-encoded antibody that catalyses the rearrangement of hexadiene 2 to aldehyde 3 with a rate approaching that of a related pericyclic reaction catalysed by the enzyme chorismate mutase. Affinity maturation gives antibody AZ-28, which has six amino acid substitutions, one of which results in a decrease in catalytic rate. To understand the relationship between binding and catalytic rate in this system we characterized a series of active-site mutants and determined the three-dimensional crystal structure of the complex of AZ-28 with the transition state analogue. This analysis indicates that the activation energy depends on a complex balance of several stereoelectronic effects which are controlled by an extensive network of binding interactions in the active site. Thus in this instance the combinatorial diversity of the immune system provided both an efficient catalyst for a reaction where no enzyme is known, as well as an opportunity to explore the mechanisms and evolution of biological catalysis.

Aldehydes↗
PubMed2004

Para-position derivatives of fungal anthelmintic cyclodepsipeptides engineered with Streptomyces venezuelae antibiotic biosynthetic genes.

PF1022A, a cyclooctadepsipeptide possessing strong anthelmintic properties and produced by the filamentous fungus Rosellinia sp. PF1022, consists of four alternating residues of N-methyl-L-leucine and four residues of D-lactate or D-phenyllactate. PF1022A derivatives obtained through modification of their benzene ring at the para-position with nitro or amino groups act as valuable starting materials for the synthesis of compounds with improved anthelmintic activities. Here we describe the production of such derivatives by fermentation through metabolic engineering of the PF1022A biosynthetic pathway in Rosellinia sp. PF1022. Three genes cloned from Streptomyces venezuelae, and required for the biosynthesis of p-aminophenylpyruvate from chorismate in the chloramphenicol biosynthetic pathway, were expressed in a chorismate mutase-deficient strain derived from Rosellinia sp. PF1022. Liquid chromatography-mass spectrometry and NMR analyses confirmed that this approach facilitated the production of PF1022A derivatives specifically modified at the para-position. This fermentation method is environmentally safe and can be used for the industrial scale production of PF1022A derivatives.

Animals↗
PubMed2002

Flavoenzymes that catalyse reactions with no net redox change.

This review covers unusual flavoenzymes that catalyse reactions with no net redox change. Some of these enzymes utilise the redox properties of flavin directly in catalysis with either two-electron chemistry (N-methylglutamate synthase and 5-hydroxyvaleryl-CoA dehydratase) or free radical chemistry (chorismate synthase, DNA photolyase, (6-4) photolyase and 4-hydroxybutyryl-CoA dehydratase). Whether the flavin has a redox role in some other flavoproteins is not yet clear ((R)-2-hydroxyacyl-CoA dehydratases, isopentenyl diphosphate isomerase and UDPgalactopyranose mutase). The remaining flavoenzymes do not make use of the redox properties of the flavin (acetohydroxyacid synthases and hydroxynitrile lyase). The literature is reviewed up to early 2002 and 121 references are cited.

Catalysis↗